Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway

ABSTRACT

Cyclic-GMP-AMP synthase (cGAS) and cyclic-GMP-AMP (cGAMP), including 2′3-cGAMP, 2′2-cGAMP, 3′2′-cGAMP and 3′3′-GAMP, are used in pharmaceutical formulations (including vaccine adjuvants), drug screens, therapies, and diagnostics.

This invention was made with government support under Grant Numbers ROIAI-093967 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

INTRODUCTION

Cytosolic DNA induces type-I interferons and other cytokines that areimportant for antimicrobial defense but can also result in autoimmunity.This DNA signaling pathway requires the adaptor protein STING and thetranscription factor IRF3, but the mechanism of DNA sensing is unclear.Here we report that mammalian cytosolic extracts synthesizedcyclic-GMP-AMP (cGAMP) in vitro from ATP and GTP in the presence of DNAbut not RNA. DNA transfection or DNA virus infection of mammalian cellsalso triggered cGAMP production. cGAMP bound to STING, leading to theactivation of IRF3 and induction of interferon-β (IFNβ). Thus, cGAMPrepresents the first cyclic di-nucleotide in metazoa and it functions asan endogenous second messenger that triggers interferon production inresponse to cytosolic DNA.

Through biochemical fractionation and quantitative mass spectrometry, wealso identified a cGAMP synthase (cGAS), which belongs to thenucleotidyltransferase family. Overexpression of cGAS activated thetranscription factor IRF3 and induced IFNβ in a STING-dependent manner.Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNAtransfection or DNA virus infection. cGAS bound to DNA in the cytoplasmand catalyzed cGAMP synthesis. These results indicate that cGAS is acytosolic DNA sensor that induces interferons by producing the secondmessenger cGAMP.

The invention applies these findings to novel methods and compositionrelating to cyclic-GMP-AMP synthase (cGAS) and cyclic-GMP-AMP (cGAMP),including their use in formulations (including vaccine adjuvants), drugscreens, therapies, and diagnostics.

SUMMARY

In one aspect the invention provides cell-based drug screens includingmethods of inhibiting cGAS, comprising contacting a cell or cell extractwith an effective amount of an exogenous cGAS inhibitor, and detecting aresultant inhibition of the synthase. In particular embodiments theresultant inhibition is detected inferentially by cyclic-GMP-AMP-inducedIRF3 activation (dimerization or nuclear translocation), interferonproduction, or NF-kB activation.

In another aspect the invention provides therapies including methods ofinhibiting cGAS, comprising contacting a cell determined to be in needthereof with an effective amount of an exogenous cGAS inhibitor. Inparticular embodiments the method comprises administering the inhibitorto a mammal determined to be in need thereof and comprising the cell,and/or the inhibitor is a small-molecule cyclase inhibitor or is acGAS-specific shRNA or siRNA.

In another aspect the invention provides in vitro drug screens includingmethods of inhibiting cGAS, comprising contacting a mixture comprisingthe synthase, ATP, GTP, and an inhibitor, under conditions wherein theinhibitor inhibits catalytic conversion by the synthase of the ATP andGTP to cyclic-GMP-AMP and inorganic pyrophosphate, and detecting aresultant inhibition of the synthase. In a particular embodiment mixturefurther comprises DNA and the conversion is DNA-dependent. In otherembodiments the cGAS is constitutively active.

In another aspect the invention provides in vitro drug binding assaysincluding methods of inhibiting cGAS binding to a substrate or cofactor,comprising contacting a mixture comprising the synthase and an ATP orGTP substrate or a DNA cofactor, and an inhibitor, under conditionswherein the inhibitor inhibits binding of the synthase to the substrateor cofactor, and detecting a resultant inhibition of the binding.

In another aspect the invention provides methods of making cGAMPcomprising forming a mixture comprising the cGAS, ATP and GTP, underconditions wherein the synthase catalytic converts the ATP and GTP tocGAMP, wherein the synthase, ATP and GTP are in predefined amounts, orthe method further comprises the step of isolating or detecting theresultant cGAMP. In particular embodiments the mixture further comprisesDNA and the conversion is DNA-dependent.

In another aspect the invention provides methods of detecting cGAMPlevels, cGAS levels or cGAS mutations comprising the step of: detectingin a sample from a person cGAMP levels, cGAS levels or cGAS mutations,and assigning to the person an autoimmune disease metric based on thecGAMP levels, cGAS levels or cGAS mutations; and optionallyadministering to the person a therapy for the autoimmune disease.

In another aspect the invention provides compositions comprising apredetermined amount of cGAMP, such as a vaccine further comprising animmunogen for a target pathogen, wherein the cGAMP provides an adjuvant.In particular embodiments, the composition is free of other cyclicdi-nucleotides, and/or otherwise suitable as an adjuvant or vaccine,e.g. sterile, pharmaceutically acceptable, in defined, predeterminedamounts, ratios, etc., and the compositions may be in bulk or unitdosages, quantified for individual usage. The invention also providesmethods of inducing or promoting an immune response comprisingadministering to a mammal in need thereof an effective amount of suchcompositions. In particular embodiments, the administering is mucosal(sublingual or intranasal), intramuscular or subcutaneous.

The invention includes all combinations of the recited particularembodiments. Further embodiments and the full scope of applicability ofthe invention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

DETAILED DESCRIPTION

The invention provides methods and composition relating to cGAS andcGAMP, including their use in formulations (including vaccineadjuvants), drug screens, therapies, and diagnostics.

Highlights: 2′3′-cGAMP is an endogenous second messenger produced bymammalian cells; 2′3′-cGAMP is a high affinity ligand for STING;2′3′-cGAMP is a potent inducer of type-I interferons; 2′3′-cGAMP bindinginduces conformational changes of STING.

In one aspect the invention provides cell-based drug screens includingmethods of inhibiting cGAS, comprising contacting a cell or cell extractwith an effective amount of an exogenous cGAS inhibitor, and detecting aresultant inhibition of the synthase. The synthase is typically human ormurine cGAS, and may be truncated, recombined in fusion protein, orotherwise modified to suit the assay. Typically the method is practicedin as a screening assay wherein the inhibitor is a candidate inhibitorfor analysis, which may be from a library, lead optimization, etc. Theinhibition be detected directly or inferentially, such as bycGAMP-induced IRF3 activation (dimerization or nuclear translocation),interferon production or NF-kB activation, direct detection of cGAMP andother products by, for examples, mass spectrometry, antibody-basedassays (e.g., ELISA, ALPHA, fluorescent polarization etc.). For example,IFN RNA may be measured by q-RT-PCR, and IRF3 dimerization by native gelelectrophoresis. Additional suitable readouts include measurements ofATP, GTP, and pyrophosphate (PPi).

In another aspect the invention provides therapies including methods ofinhibiting cGAS, comprising contacting a cell determined to be in needthereof with an effective amount of an exogenous cGAS inhibitor. Inparticular embodiments the method comprises administering the inhibitorto a mammal determined to be in need thereof and comprising the cell,and/or the inhibitor is a small-molecule cyclase inhibitor, or is acGAS-specific shRNA or siRNA, or other RNAi or antisense RNAcGAS-specific inhibitor.

Our data indicate that cGAS and the cGAS-cGAMP pathway is important fortriggering inflammatory responses to self and foreign DNA, and hencecGAS inhibitors can be used to reduce pathogenic cGAS activity ofassociated autoimmune diseases. Similarly, our data indicate that cGASis also important for the transformation from normal to cancer cells andalso for the survival and metastasis of cancer cells, and hence cGASinhibitors can be used to reduce pathogenic cGAS activity of associatedneoplastic diseases.

Current therapy for lupus and other autoimmune diseases involve massivedoses of immunosuppressive agents, which have severe side effects.Although a new BAFF antibody (Benlysta) has been approved for lupustreatment, it is only marginally effective. Targeting cGAS with smallmolecule inhibitors, particularly orally available ones, providessignificant advantages over the existing therapies. cGAS inhibitorstarget the root cause of lupus and other autoimmune diseases, andprovide therapeutic benefits to patients. Moreover, the cytosolic DNAinnate immunity pathway is aberrantly activated under autoimmuneconditions such as systemic lupus erythematosus (SLE), Sjögren'ssyndrome, and Aicardi-Goutibres syndrome, and cGAS inhibition provides arational treatment of these and other autoimmune diseases.

In another aspect the invention provides in vitro drug screens includingmethods of inhibiting cGAS, comprising contacting a mixture comprisingthe synthase, ATP, GTP, and an inhibitor, under conditions wherein theinhibitor inhibits catalytic conversion by the synthase of the ATP andGTP to cGAMP and inorganic pyrophosphate, and detecting a resultantinhibition of the synthase. In a particular embodiment mixture furthercomprises DNA and the conversion is DNA-dependent. In other embodimentsthe cGAS is constitutively active. Typically the method is practiced inas a screening assay wherein the inhibitor is a candidate inhibitor foranalysis, which may be from a library, lead optimization, etc. Themixture may be contained in cell or cell extract, or may be acellular.

In another aspect the invention provides in vitro drug binding assaysincluding methods of inhibiting cGAS binding to a substrate or cofactor,comprising contacting a mixture comprising the synthase and an ATP orGTP substrate or a DNA cofactor, and an inhibitor, under conditionswherein the inhibitor inhibits binding of the synthase to the substrateor cofactor, and detecting a resultant inhibition of the binding.Typically the method is practiced in as a screening assay wherein theinhibitor is a candidate inhibitor for analysis, and may be implementedin variety of suitable formats including solid phase immune assays,fluorescent polarization assays, etc.

In another aspect the invention provides methods of making cGAMPcomprising forming a mixture comprising the cGAS, ATP and GTP, underconditions wherein the synthase catalytic converts the ATP and GTP tocGAMP, wherein the synthase, ATP and GTP are in predefined amounts, orthe method further comprises the step of isolating or detecting theresultant cGAMP. In particular embodiments the mixture further comprisesDNA and the conversion is DNA-dependent.

Pathogenic expression of cGAS activity, particularly as a result ofover-expression or mutation is associated with human autoimmunediseases; hence, the invention also provides methods and assays fordetecting cGAS levels or mutations, particularly as a diagnostic toolfor human autoimmune diseases. Accordingly, in another aspect theinvention provides methods of detecting cGAMP levels, cGAS levels orcGAS mutations comprising the step of: detecting in a sample from aperson cGAMP levels, cGAS levels or cGAS mutations, and assigning to theperson an autoimmune disease metric based on the cGAMP levels, cGASlevels or cGAS mutations; and optionally administering to the person atherapy for the autoimmune disease.

In another aspect the invention provides compositions comprising apredetermined amount of cGAMP, such as a vaccine further comprising animmunogen for a target pathogen, wherein the cGAMP provides an adjuvant.In particular embodiments, the composition is substantially oressentially free of other cyclic di-nucleotides. The invention alsoprovides methods of inducing or promoting an immune response comprisingadministering to a mammal in need thereof an effective amount of suchcompositions. In particular embodiments, the administering is mucosal(sublingual or intranasal), intramuscular or subcutaneous.

As a potent inducer of type-I interferons, cGAMP provides a rationalimmune adjuvant. cGAMP may be used as vaccine adjuvants, particularlywith mucosal vaccines, and may be formulated with immunogens anddelivered as have been cyclic-di-GMP and c-di-AMP as vaccine adjuvants;see, e.g. Pedersen, et al. PLoS ONE, November 2011, 6, 11, e26973;Ebensen et al., Vaccine 29, 2011, 5210-5220; Chen et al., Vaccine 28,2010, 3080-3085. In fact the cGAMP adjuvant are often more effectivebecause cGAMP is more potent than c-di-GMP in inducing interferons.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein, including citations therein, are herebyincorporated by reference in their entirety for all purposes.

EXAMPLES Example 1. Cyclic-GMP-AMP is an Endogenous Second Messenger inInnate Immune Signaling by Cytosolic DNA

Host defense against foreign genetic elements is one of the mostfundamental functions of a living organism. The presence of self orforeign DNA in the cytoplasm is sensed by eukaryotic cells as a dangersignal or a sign of foreign invasion (1). DNA can be introduced into thecytoplasm by bacterial or viral infection, transfection, or ‘leakage’from the nucleus or mitochondria under some pathological conditions thatcause autoimmune diseases such as lupus. In mammalian cells, cytosolicDNA triggers the production of type-I interferons (IFNs) and othercytokines through the endoplasmic reticulum protein STING (also known asMITA, MPYS or ERIS) (2). STING recruits and activates the cytosolickinases IKK and TBK1, which activate the transcription factors NF-κB andIRF3, respectively. NF-κB and IRF3 then enter the nucleus and functiontogether to induce IFNs and other cytokines. DNA-dependent RNApolymerase III has been shown to be a sensor that detects andtranscribes AT-rich DNA such as poly[dA:dT] into an RNA ligand capableof stimulating the RIG-I pathway to induce IFNs (3, 4). However, mostDNA sequences do not activate the RNA polymerase III—RIG-I pathway.Instead, cytosolic DNA activates the STING-dependent pathway in asequence-independent manner. How cytosolic DNA activates the STINGpathway remains elusive.

We hypothesized that DNA binds to and activates a putative cytosolic DNAsensor, which then directly or indirectly activates STING, leading tothe activation of IRF3 and NF-xB. To test this model, we developed an invitro complementation assay using the murine fibrosarcoma cell lineL929, which is known to induce interferon-β (IFNβ) in a STING-dependentmanner (5). We used a L929 cell line stably expressing a short hairpin(sh)RNA against STING such that DNA transfection would only activatefactors upstream of STING, including the putative DNA sensor. TheL929-shSTING cells were transfected with different types of DNA and thencytoplasmic extracts from these cells were mixed with the humanmonocytic cell line THP1 or murine macrophage cell line Raw264.7, whichwas permeabilized with perfringolysin O (PFO). PFO treatment pokes holesin the plasma membrane (6), allowing the cytoplasm to diffuse in and outof cells, while retaining organelles including endoplasmic reticulum(which contains STING) and Golgi apparatus inside the cells (7). If anupstream activator of STING is generated in the DNA transfected cells,the cytoplasm containing such an activator is expected to activate STINGin the PFO-permeabilized cells, leading to the phosphorylation anddimerization of IRF3.

Cytoplasmic extracts from L929-shSTING cells transfected with a DNAsequence known as interferon-stimulatory DNA (ISD), poly[dA:dT], aGC-rich 50-base pair dsDNA (G:C50), poly[dI:dC] or herring testis DNA(HT-DNA) activated IRF3 in permeabilized THP1 cells, indicating thatthis activity was independent of DNA sequence.

To determine if the STING activator is a protein, we incubated thecytoplasmic extracts at 95° C. to denature most proteins and thenincubated the ‘heat supernatant’ with permeabilized THP1 cells.Surprisingly, the heat supernatant from the ISD or HT DNA transfectedcells caused IRF3 dimerization. This activity was resistant to treatmentwith Benzonase, which degrades both DNA and RNA, or proteinase K. Thus,the STING activator is probably not a protein, DNA or RNA.

To test if DNA could stimulate the generation of the heat-resistantSTING activator in vitro, we incubated HT DNA with L929-shSTINGcytoplasmic extracts (S100) in the presence of ATP. The reaction mixturewas heated at 95° C. to denature proteins. Remarkably, incubation of thesupernatant with permeabilized Raw264.7 cells led to IRF3 dimerization.This activity depended on the addition of DNA to the cytoplasmicextracts. Other DNA, including poly[dA:dT], poly[dG:dC] and ISD, alsostimulated the generation of the STING activator in L929-shSTINGcytoplasmic extracts, whereas poly[I:C] and single-stranded RNA had noactivity. Similar results were obtained with permeabilized THP1 cells.Knockdown of STING in the permeabilized THP1 cells abolished IRF3activation by the heat-resistant factor generated by DNA transfectedinto L929 cells or DNA added to L929 cytosolic extracts. Controlexperiments showed that the knockdown of STING inhibited the activationof IRF3 and induction of IFNβ and TNFα in THP1 cells by HT-DNAtransfection, but IRF3 activation by poly[I:C] transfection or Sendaivirus infection, which is known to activate the RIG-I pathway, wasunaffected by the STING knockdown. We also tested cytoplasmic extractsfrom several cell lines for their ability to produce the heat-resistantSTING activator. Incubation of HT-DNA with extracts from primary MEFcells, mouse bone marrow derived macrophages (BMDM) and L929 cells ledto generation of the heat-resistant factor that activated IRF3. Humancell extracts from THP1, but not HEK293T, were also able to produce thisSTING activator. These results are in agreement with our previousfinding that primary MEF, BMDM, L929 and THP1 cells, but not HEK293Tcells, possessed the STING-dependent, RNA polymerase III-independent,pathway to induce type-I interferons (3).

We next purified the heat-resistant STING activator from L929 cellextracts using several chromatographic steps including a STING-Flagaffinity purification step. Previous research has shown that thebacterial molecules cyclic-di-AMP and cyclic-di-GMP bind to STING andinduce type-I interferons (8, 9). However, using nano liquidchromatography mass spectrometry (nano-LC-MS), we did not detect MS orMS/MS spectra consistent with those expected of c-di-GMP ([M+H]+=691) orc-di-AMP ([M+H]+=659). Interestingly, in-depth examination of the MSspectra revealed two ions with mass to charge ratios (m/z) of 675.1(z=1+) and 338.1 (z=2+), which were present in the active fractions butabsent in the background spectra. These m/z values, despite the low massaccuracy of the mass spectrometer (LTQ), were equivalent to the averagecalculated m/z values of c-di-GMP and c-di-AMP (675=[691+659]/2). Thisobservation indicated that the detected ion was a hybrid of c-di-GMP andc-di-AMP, i.e., cyclic-GMP-AMP (m/z=675.107, z=1+; m/z=338.057, z=2+).Collision induced dissociation (CID) fragmentation of this ion(m/z=338.1, z=2⁺⁾revealed several prominent ions with m/z valuesexpected of the product ions of c-GMP-AMP (cGAMP). Importantly,quantitative mass spectrometry using selective reaction monitoring (SRM)showed that the abundance of the ions representing cGAMP in thefractions from a C18 column correlated very well with theirIRF3-stimulatory activities. cGAMP has recently been identified in thebacterium Vibro cholera and shown to play a role in bacterial chemotaxisand colonization (10). However, cGAMP has not been reported to exist orfunction in eukaryotic cells.

To verify the identity of the heat resistant STING activator, we used ahigh-resolution high-accuracy mass spectrometer (Q Exactive, Thermo) toperform nano-LC-MS analysis. The cell-derived STING activator had m/z of675.107 (z=1+) and 338.057 (z=2+), which exactly matched the theoreticalvalues of cGAMP. To further characterize the structure and function ofcGAMP, we developed a ten-step single-flask protocol to chemicallysynthesize cGAMP. The MS/MS spectra of the cell-derived STING activatorwere identical to those of the chemically synthesized cGAMP. Theseresults demonstrate that L929 cells produced cGAMP.

Quantitative RT-PCR and ELISA assays showed that chemically synthesizedcGAMP induced IFNβ RNA and protein in L929 cells after introduction intothe cells. Titration experiments showed that cGAMP induced IFNβ RNArobustly even at concentrations as low as 10 nM. In fact, cGAMP was muchmore potent than c-di-GMP in inducing IFNβ based on ELISA assays. cGAMPwas also more potent than c-di-GMP and c-di-AMP in activating IRF3. Todetermine if L929 extracts contained enzymes that could synthesize othertypes of di-nucleotides or oligonucleotides capable of activating IRF3,we tested all four ribonucleotides in various combinations. ATP and GTPwere both necessary and sufficient to support the synthesis of anactivator of IRF3, further supporting that L929 contained an enzyme thatsynthesizes cGAMP from ATP and GTP.

To determine if DNA virus infection leads to the production of cGAMP incells, we infected L929 cells with HSV-1 lacking ICP34.5, a viralprotein known to antagonize interferon production in the infected cells(11). Like DNA transfection, HSV-1AICP34.5 infection led to IRF3activation in L929 cells. Cell extracts from the DNA-transfected orvirus-infected cells contained a heat-resistant factor that couldactivate IRF3 in permeabilized Raw264.7 cells. As a control, we infectedL929 cells with a vesicular stomatitis virus strain, VSV-AM51-GFP, anRNA virus known to trigger strong interferon production through theRIG-I pathway (12, 13). In contrast to HSV-1, VSV-infected cells did notcontain the heat-resistant IRF3 activator in the same in vitro assay,although VSV infection did induce IRF3 activation in L929 cells. Theheat resistant factor in HSV-1 infected cells was enriched by reversephase HPLC and quantified by nano-LC-MS using SRM. DNA transfected orHSV-1 infected cells, but not mock treated or VSV infected cells,produced elevated levels of cGAMP. Kinetic experiments showed that,after DNA was transfected into L929 cells, cGAMP was produced beforeIRF3 dimerization, and IFNβ induction could be detected. To test if DNAviruses could induce cGAMP production in human cells, we infected THP1cells with HSV1 or Vaccinia virus (VACV). Both viruses induced IRF3dimerization in the cells. Importantly, both viruses also triggered theproduction of cGAMP that activated IRF3. Collectively, these resultsindicate that DNA transfection and DNA virus infections in human andmouse cells produced cGAMP, which led to IRF3 activation.

To determine if cGAMP activates IRF3 through STING, we carried out threesets of experiments. First, we established a HEK293T cell line stablyexpressing STING, stimulated these cells with cGAMP and then measuredIFNβ induction by quantitative RT-PCR. HEK293T cells did not respond tocGAMP, likely due to a lack of or a very low level of STING expressionin these cells. The expression of STING in HEK293T cells rendered a highlevel of IFNβ induction by cGAMP. However, DNA did not stimulateHEK293T/STING cells to induce IFNβ, consistent with a defect of HEK293Tcells in producing cGAMP in response to DNA stimulation. In contrast,L929 cells induced IFNβ in response to stimulation by either cGAMP orDNA. HSV-1 infection induced IRF3 dimerization in L929, but not HEK293Tor HEK29T/STING cells, indicating that the production of cGAMP isimportant for HSV-1 to activate IRF3 in cells. Indeed, extracts fromHSV1-infected L929, but not from HEK293T or HEK293T/STING cells,contained the cGAMP activity that led to IRF3 dimerization inpermeabilized Raw264.7 cells. These results indicate that the expressionof STING in HEK293T cells installed the ability of the cells to activateIRF3 and induce IFNβ in response to cGAMP, but was insufficient toinstall the response to DNA or DNA viruses due to a defect of HEK293Tcells in synthesizing cGAMP.

Second, we tested the response of L929 and L929-shSTING cells to cGAMP.Similar to ISD and c-di-GMP, cGAMP-induced IRF3 dimerization wasdependent on STING. In contrast, poly[I:C] still induced IRF3dimerization in the absence of STING. These results demonstrate thatSTING is necessary for cGAMP to activate IRF3.

Finally, we examined whether STING binds to cGAMP directly. RecombinantSTING protein containing residues 139-379, which has been shown to bindc-di-GMP (14), was expressed and purified from E. coli and thenincubated with ³²P-cGAMP followed by UV-induced crosslinking. Aradiolabelled band corresponding to cross-linked STING-cGAMP complex wasdetected when both STING and ³²P-cGAMP were present. High concentrationsof ATP or GTP did not compete with the formation of STING-cGAMP complex.By contrast, the intensity of this band decreased as the concentrationsof competing cold cGAMP, c-di-GMP or c-di-AMP increased, indicating thatthe cGAMP binding sites on STING overlap with those that interact withc-di-GMP and c-di-AMP. Indeed, mutations of several residues that wererecently shown to participate in the binding of STING to c-di-GMP (14),including S161Y, Y240S and N242A, also impaired the binding of STING tocGAMP. Collectively, these results demonstrate that cGAMP is a ligandthat binds to and activates STING.

Cyclic di-nucleotides have been shown to function as bacterial secondmessengers that regulate a variety of physiological processes, includingbacterial motility and biofilm formation (15). A recent report showedthat c-di-GMP is produced in the protozoan Dictyostelium and functionsas a morphogen to induce stalk cell differentiation (16). In thisexample, we identified cGAMP as the first cyclic di-nucleotide inmetazoa. Moreover, we showed that cGAMP is a potent inducer of type-Iinterferons. The role of cGAMP is similar to that of cAMP, thebest-studied second messenger (17). Like cAMP, which is synthesized byadenylate cyclase upon its activation by upstream ligands, cGAMP issynthesized by a cyclase in response to stimulation by a DNA ligand(18). cAMP binds to and activates protein kinase A and other effectormolecules. Similarly, cGAMP binds to and activates STING to trigger thedownstream signaling cascades. As an endogenous molecule in mammaliancells, cGAMP may be used in immune therapy or as a vaccine adjuvant.

REFERENCES AND NOTES

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Example 2. Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor thatActivates the Type-I Interferon Pathway

DNA was known to stimulate immune responses long before it was shown tobe a genetic material, but the mechanism by which DNA functions as animmune stimulant remains poorly understood (1). Although DNA canstimulate the production of type-I interferons in dendritic cellsthrough binding to Toll-like receptor 9 (TLR9) in the endosome, how DNAin the cytosol induces IFN is still unclear. In particular, the sensorthat detects cytosolic DNA in the interferon pathway remains elusive(2). Although several proteins, including DAI, RNA polymerase III,IF116, DDX41 and several other DNA helicases, have been suggested tofunction as the potential DNA sensors that induce IFN, none has been metwith universal acceptance (3).

Purification and Identification of Cyclic GMP-AMP Synthase (cGAS).

We showed that delivery of DNA to mammalian cells or cytosolic extractstriggered the production of cyclic GMP-AMP (cGAMP), which bound to andactivated STING, leading to the activation of IRF3 and induction of IFNβ(4). To identify the cGAMP synthase (cGAS), we fractionated cytosolicextracts (S100) from the murine fibrosarcoma cell line L929, whichcontains the cGAMP synthesizing activity. This activity was assayed byincubating the column fractions with ATP and GTP in the presence ofherring testis DNA (HT-DNA). After digesting the DNA with Benzonase andheating at 95° C. to denature proteins, the heat-resistant supernatantsthat contained cGAMP were incubated with Perfringolysin O(PFO)-permeabilized Raw264.7 cells (transformed mouse macrophages).cGAMP-induced IRF3 dimerization in these cells were analyzed by nativegel electrophoresis (4). Using this assay, we carried out threeindependent routes of purification, each consisting of four steps ofchromatography but differing in the columns or the order of the columnsthat were used. In particular, the third route included an affinitypurification step using a biotinylated DNA oligo (a 45-bp DNA known asImmune Stimulatory DNA or ISD). We estimated that we achieved a range of8000-15,000 fold purification and 2-5% recovery of the activity fromthese routes of fractionation. However, in the last step of each ofthese purification routes, silver staining of the fractions did notreveal clear protein bands that co-purified with the cGAS activity,suggesting that the abundance of the putative cGAS protein might be verylow in L929 cytosolic extracts.

We developed a quantitative mass spectrometry strategy to identify alist of proteins that co-purified with the cGAS activity at the laststep of each purification route. We reasoned that the putative cGASprotein must co-purify with its activity in all three purificationroutes, whereas most ‘contaminating’ proteins would not. Thus, from thelast step of each purification route, we chose fractions that containedmost of the cGAS activity (peak fractions) and adjacent fractions thatcontained very weak or no activity. The proteins in each fraction wereseparated by SDS-PAGE and identified by nano liquid chromatography massspectrometry (nano-LC-MS). The data were analyzed by label-freequantification using the MaxQuant software (5). Remarkably, althoughmany proteins co-purified with the cGAS activity in one or twopurification routes, only three proteins co-purified in all threeroutes. All three were putative uncharacterized proteins: E330016A19(accession #: NP_775562), Arf-GAP with dual PH domain-containing protein2 (NP_742145) and signal recognition particle 9 kDa protein (NP_036188).Among these, more than 24 unique peptides were identified in E330016A19,representing 41% coverage in this protein of 507 amino acids.

Bioinformatic analysis drew our attention to E330016A19, which exhibitedstructural and sequence homology to the catalytic domain ofoligoadenylate synthase (OAS1). In particular, E330016A19 contains aconserved G[G/S]x₉-1₃[E/D]h[E/D]h motif, where x₉-1₃ indicates 9-13flanking residues consisting of any amino acid and h indicates ahydrophobic amino acid. This motif is found in thenucleotidyltransferase (NTase) family (6). Besides OAS1, this familyincludes adenylate cyclase, poly[A] polymerase and DNA polymerases. TheC-terminus of E330016A19 contained a Male Abnormal 21 (Mab21) domain,which was first identified in the C. elegans protein Mab21 (7). Sequencealignment revealed that the C-terminal NTase and Mab21 domains arehighly conserved from zebrafish to human, whereas the N-terminalsequences are much less conserved (8). Interestingly, the humanhomologue of E330016A19, C6orf150 (also known as MB21D1) was recentlyidentified as one of several positive hits in a screen forinterferon-stimulated genes (ISGs) whose overexpression inhibited viralreplication (9). For clarity and on the basis of evidence presented inthis paper, we propose to name the mouse protein E330016A19 as m-cGASand the human homologue C6orf150 as h-cGAS. Quantitative RT-PCR showedthat the expression of m-cGAS was low in immortalized MEF cells but highin L929, Raw264.7 and bone marrow-derived macrophages (BMDM). Similarly,the expression of h-cGAS RNA was very low in HEK293T cells but high inthe human monocytic cell line THP1. Immunoblotting further confirmedthat h-cGAS protein was expressed in THP1 but not HEK293T cells. Thus,the expression levels of m-cGAS and h-cGAS in different cell linescorrelated with the ability of these cells to produce cGAMP and induceIFNβ in response to cytosolic DNA (4, 10).

Catalysis by cGAS Triggers Type-I Interferon Production.

Overexpression of m-cGAS in HEK293T, which lacks STING expression, didnot induce IFNβ, whereas stable expression of STING in HEK293T cellsrendered these cells highly competent in IFNβ induction by m-cGAS.Importantly, point mutations of the putative catalytic residues G198 andS199 to alanine abolished the ability of m-cGAS to induce IFNβ. Thesemutations, as well as mutations of the other putative catalytic residuesE211 and D213 to alanine, also abrogated the ability of m-cGAS to induceIRF3 dimerization in HEK293T-STING cells. The magnitude of IFNβinduction by c-GAS was comparable to that induced by MAVS (an adaptorprotein that functions downstream of the RNA sensor RIG-I) and wasseveral orders higher than those induced by other putative DNA sensors,including DAI, IFI16 and DDX41. To determine if overexpression of cGASand other putative DNA sensors led to the production of cGAMP in cells,supernatants from heat-treated cell extracts were incubated withPFO-permeabilized Raw264.7 cells, followed by measurement of IRF3dimerization. Among all the proteins expressed in HEK293T-STING cells,only cGAS was capable of producing the cGAMP activity in the cells.

To test if cGAS could synthesize cGAMP in vitro, we purified wild-type(WT) and mutant Flag-cGAS proteins from transfected HEK293T cells. WTm-cGAS and h-cGAS, but not the catalytically inactive mutants of cGAS,were able to produce the cGAMP activity, which stimulated IRF3dimerization in permeabilized Raw264.7 cells. Importantly, the in vitroactivities of both m-cGAS and h-cGAS were dependent on the presence ofHT-DNA. To test if DNA enhances IFNβ induction by cGAS in cells,different amounts of cGAS expression plasmid was transfected with orwithout HT-DNA into HEK293T-STING cells. HT-DNA significantly enhancedIFNβ induction by low (10 and 50 ng) but not high (200 ng) doses of cGASplasmid. In contrast to cGAS, IFI16 and DDX41 did not induce IFNβ evenwhen HT-DNA was co-transfected.

cGAS is Required for IFNβ Induction by DNA Transfection and DNA VirusInfection.

We used two different pairs of siRNA to knock down m-cGAS in L929 cells,and found that both siRNA oligos significantly inhibited IFNβ inductionby HT-DNA, and that the degree of inhibition correlated with theefficiency of knocking down m-cGAS RNA. We also established two L929cell lines stably expressing shRNA sequences targeting distinct regionsof m-cGAS. The ability of these cells to induce IFNβ in response toHT-DNA was severely compromised as compared to another cell lineexpressing a control shRNA (GFP). Importantly, expression of cGAS in theL929-sh-cGAS cells restored IFNβ induction. Expression of STING or MAVSin L929-sh-cGAS cells or delivery of cGAMP to these cells also inducedIFNβ. In contrast, expression of cGAS or delivery of cGAMP failed toinduce IFNβ in L929-shSTING cells, whereas expression of STING or MAVSrestored IFNβ induction in these cells. Quantitative RT-PCR analysesconfirmed the specificity and efficiency of knocking down cGAS and STINGin the L929 cell lines stably expressing the corresponding shRNAs. Theseresults indicate that cGAS functions upstream of STING and is requiredfor IFNβ induction by cytosolic DNA.

Herpes simplex virus 1 (HSV-1) is a DNA virus known to induce IFNsthrough the activation of STING and IRF3 (3). Importantly, shRNA againstm-cGAS, but not GFP, in L929 cells strongly inhibited IRF3 dimerizationinduced by HSV-1 infection. In contrast, knockdown of cGAS did notaffect IRF3 activation by Sendai virus, an RNA virus. To determine ifcGAS is required for the generation of cGAMP in cells, we transfectedHT-DNA into L929-shGFP and L929-sh-cGAS or infected these cells withHSV-1, then prepared heat-resistant fractions that contained cGAMP,which was subsequently delivered to permeabilized Raw264.7 cells tomeasure IRF3 activation. Knockdown of cGAS largely abolished the cGAMPactivity generated by DNA transfection or HSV-1 infection. Quantitativemass spectrometry using selective reaction monitoring (SRM) showed thatthe abundance of cGAMP induced by DNA transfection or HSV-1 infectionwas markedly reduced in L929 cells depleted of cGAS. Taken together,these results demonstrate that cGAS is essential for producing cGAMP andactivating IRF3 in response to DNA transfection or HSV-1 infection.

To determine if cGAS is important in the DNA sensing pathway in humancells, we established a THP1 cell line stably expressing a shRNAtargeting h-cGAS. The knockdown of h-cGAS strongly inhibited IFNβinduction by HT-DNA transfection or infection by vaccinia virus, anotherDNA virus, but not Sendai virus. The knockdown of h-cGAS also inhibitedIRF3 dimerization induced by HSV-1 infection in THP1 cells. This resultwas further confirmed in another THP1 cell line expressing a shRNAtargeting a different region of h-cGAS. The strong and specific effectsof multiple cGAS shRNA sequences in inhibiting DNA-induced IRF3activation and IFNβ induction in both mouse and human cell linesdemonstrate a key role of cGAS in the STING-dependent DNA sensingpathway.

Recombinant cGAS Protein Catalyzes cGAMP Synthesis from ATP and GTP in aDNA-Dependent Manner.

To test if cGAS is sufficient to catalyze cGAMP synthesis, we expressedFlag-tagged h-cGAS in HEK293T cells and purified it to apparenthomogeneity. In the presence of HT-DNA, purified c-GAS protein catalyzedthe production of cGAMP activity, which stimulated IRF3 dimerization inpermeabilized Raw264.7 cells. DNase-I treatment abolished this activity.The cGAS activity was also stimulated by other DNA, includingpoly(dA:dT), poly(dG:dC) and ISD, but not the RNA poly(I:C). Thesynthesis of cGAMP by cGAS required both ATP and GTP, but not CTP orUTP. These results indicate that the cyclase activity of purified cGASprotein was stimulated by DNA but not RNA.

We also expressed m-cGAS in E. coli as a SUMO fusion protein. Afterpurification, Sumo-m-cGAS generated the cGAMP activity in aDNA-dependent manner. However, after the SUMO tag was removed by a Sumoprotease, the m-cGAS protein catalyzed cGAMP synthesis in aDNA-independent manner. The reason for this loss of DNA dependency isunclear, but could be due to some conformational changes after Sumoremoval. Titration experiments showed that less than 1 nM of therecombinant cGAS protein led to detectable IRF3 dimerization, whereasthe catalytically inactive mutant of cGAS failed to activate IRF3 evenat high concentrations. To formally prove that cGAS catalyzes thesynthesis of cGAMP, the reaction products were analyzed by nano-LC-MSusing SRM. cGAMP was detected in a 60-min reaction containing purifiedcGAS, ATP and GTP. The identity of cGAMP was further confirmed by ionfragmentation using collision-induced dissociation (CID). Thefragmentation pattern of cGAMP synthesized by purified cGAS revealedproduct ions whose m/z values matched those of chemically synthesizedcGAMP. Collectively, these results demonstrate that purified cGAScatalyzes the synthesis of cGAMP from ATP and GTP.

cGAS binds to DNA. The stimulation of cGAS activity by DNA indicatesthat c-GAS is a DNA sensor. Indeed, both GST-m-cGAS and GST-h-cGAS, butnot GST-RIG-I N-terminus [RIG-I(N)], were precipitated by biotinylatedISD. In contrast, biotinylated RNA did not bind cGAS. Deletion analysesshowed that the h-cGAS N-terminal fragment containing residues 1-212,but not the C-terminal fragment 213-522, bound to ISD. A longerC-terminal fragment containing residues 161-522 did bind to ISD,indicating that the sequence 161-212 may be important for DNA binding.However, deletion of residues 161-212 from h-cGAS did not significantlyimpair ISD binding, indicating that cGAS contains another DNA bindingdomain at the N-terminus. Indeed, the N-terminal fragment containingresidues 1-160 also bound ISD. Thus, cGAS may contain two separate DNAbinding domains at the N-terminus. Nevertheless, it is clear that theN-terminus of h-cGAS containing residues 1-212 is both necessary andsufficient to bind DNA.

Different deletion mutants of h-cGAS were overexpressed in HEK293T-STINGcells to determine their ability to activate IRF3 and induce IFNβ andthe cytokine tumor necrosis factor α (TNFα). The protein fragment 1-382,which lacks the C-terminal 140 residues including much of the Mab21domain, failed to induce IFNβ or TNFα or to activate IRF3, indicatingthat an intact Mab21 domain is important for cGAS function. As expected,deletion of the N-terminal 212 residues (fragment 213-522), whichinclude part of the NTase domain, abolished the cGAS activity. Aninternal deletion of just four amino acids (KLKL, A171-174) within thefirst helix of the NTase fold preceding the catalytic residues alsodestroyed the cGAS activity. Interestingly, deletion of the N-terminal160 residues did not affect IRF3 activation or cytokine induction bycGAS. In vitro assay showed that this protein fragment (161-522) stillactivated the IRF3 pathway in a DNA-dependent manner. Thus, theN-terminal 160 amino acids of h-cGAS, whose primary sequence is nothighly conserved evolutionarily, appears to be largely dispensable forDNA binding and catalysis by cGAS. In contrast, the NTase and Mab21domains are important for cGAS activity.

cGAS is Predominantly Localized in the Cytosol.

To determine if cGAS is a cytosolic DNA sensor, we prepared cytosolicand nuclear extracts from THP1 cells and analyzed the localization ofendogenous h-cGAS by immunoblotting. h-cGAS was detected in thecytosolic extracts, but barely detectable in the nuclear extracts. TheTHP1 extracts were further subjected to differential centrifugation toseparate subcellular organelles from one another and from the cytosol.Similar amounts of h-cGAS were detected in S100 and P100 (pellet after100,000×g centrifugation), indicating that this protein is soluble inthe cytoplasm but a significant fraction of the protein is associatedwith light vesicles or organelles. The cGAS protein was not detected inP5, which contained mitochondria and ER as evidenced by the presence ofVDAC and STING, respectively. cGAS was also not detectable in P20, whichcontained predominantly ER and heavy vesicles.

We also examined the localization of cGAS by confocal immunofluorescencemicroscopy using L929 cells stably expressing Flag-m-cGAS. The cGASprotein distributed throughout the cytoplasm but could also be observedin the nuclear or peri-nuclear region. Interestingly, after the cellswere transfected with Cy3-labelled ISD for 2 or 4 hours, punctate formsof cGAS were observed and they overlapped with the DNA fluorescence.Such co-localization and apparent aggregation of cGAS and Cy3-ISD wasobserved in more than 50% of the cells under observation. These results,together with the biochemical evidence of direct binding of cGAS withDNA, indicate that cGAS binds to DNA in the cytoplasm.

Discussion.

In this example, we developed a strategy that combined quantitative massspectrometry with conventional protein purification to identifybiologically active proteins that were partially purified from crudecell extracts. This strategy is generally applicable to proteins thatare difficult to be purified to homogeneity due to very low abundance,labile activity or scarce starting materials. As a proof of principle,we used this strategy to identify the mouse protein E330016A19 as theenzyme that synthesizes cGAMP. This discovery led to the identificationof a large family of cGAS that is conserved from fish to human, formallydemonstrating that vertebrate animals contain evolutionarily conservedenzymes that synthesize cyclic di-nucleotides, which were previouslyfound only in bacteria, archaea and protozoan (11-13). Vibrio choleracan synthesize cGAMP through its cyclase DncV (VC0179), which containsan NTase domain, but lacks significant primary sequence homology to themammalian cGAS (12).

Our results not only demonstrate that cGAS is a cytosolic DNA sensorthat triggers the type-I interferon pathway, but also reveal a novelmechanism of immune signaling in which cGAS generates the secondmessenger cGAMP, which binds to and activates STING (4), therebytriggering type-I interferon production. The deployment of cGAS as acytosolic DNA sensor greatly expands the repertoire of microorganismsdetected by the host immune system. In principle, all microorganismsthat can carry DNA into the host cytoplasm, such as DNA viruses,bacteria, parasites (e.g., malaria) and retroviruses (e.g., HIV), couldtrigger the cGAS-STING pathway (14, 15). The enzymatic synthesis ofcGAMP by cGAS provides a mechanism of signal amplification for a robustand sensitive immune response. However, the detection of self DNA in thehost cytoplasm by cGAS can also lead to autoimmune diseases, such assystemic lupus erythematosus, Sjögren's syndrome, and Aicardi-Goutibressyndrome (16-18).

Several other DNA sensors, such as DAI, IFI16 and DDX41, have beenreported to induce type-I interferons (19-21). Overexpression of DAI,IFI16 or DDX41 did not lead to the production of cGAMP. We also foundthat knockdown of DDX41 and p204 (a mouse homologue of IFI16) by siRNAdid not inhibit the generation of cGAMP activity in HT-DNA transfectedL929 cells. Unlike other putative DNA sensors and most patternrecognition receptors (e.g., TLRs), cGAS is a cyclase that is amenableto inhibition by small molecule compounds, which provide therapeuticagents for the treatment of human autoimmune diseases.

REFERENCES AND NOTES

-   1. L. A. O'Neill, DNA makes RNA makes innate immunity. Cell 138, 428    (Aug. 7, 2009).-   2. G. N. Barber, Cytoplasmic DNA innate immune pathways.    Immunological reviews 243, 99 (September, 2011).-   3. S. E. Keating, M. Baran, A. G. Bowie, Cytosolic DNA sensors    regulating type I interferon induction. Trends in immunology 32, 574    (December, 2011).-   4. J. Wu et al., Cyclic-GMP-AMP is an endogenous second messenger in    innate immune signaling by cytosolic DNA. Science, (2012).-   5. J. Cox, M. Mann, MaxQuant enables high peptide identification    rates, individualized p.p.b.-range mass accuracies and proteome-wide    protein quantification. Nat Biotechnol 26, 1367 (December, 2008).-   6. K. Kuchta, L. Knizewski, L. S. Wyrwicz, L. Rychlewski, K.    Ginalski, Comprehensive classification of nucleotidyltransferase    fold proteins: identification of novel families and their    representatives in human. Nucleic Acids Res 37, 7701 (December,    2009).-   7. K. L. Chow, D. H. Hall, S. W. Emmons, The mab-21 gene of    Caenorhabditis elegans encodes a novel protein required for choice    of alternate cell fates. Development 121, 3615 (November, 1995).-   8. J. Pei, B. H. Kim, N. V. Grishin, PROMALS3D: a tool for multiple    protein sequence and structure alignments. Nucleic Acids Res 36,    2295 (April, 2008).-   9. J. W. Schoggins et al., A diverse range of gene products are    effectors of the type I interferon antiviral response. Nature 472,    481 (Apr. 28, 2011).-   10. Y. H. Chiu, J. B. Macmillan, Z. J. Chen, RNA polymerase III    detects cytosolic DNA and induces type I interferons through the    RIG-I pathway. Cell 138, 576 (Aug. 7, 2009).-   11. C. Pesavento, R. Hengge, Bacterial nucleotide-based second    messengers. Curr Opin Microbiol 12, 170 (April, 2009).-   12. B. W. Davies, R. W. Bogard, T. S. Young, J. J. Mekalanos,    Coordinated regulation of accessory genetic elements produces cyclic    di-nucleotides for V. cholerae virulence. Cell 149, 358 (Apr. 13,    2012).-   13. Z. H. Chen, P. Schaap, The prokaryote messenger c-di-GMP    triggers stalk cell differentiation in Dictyostelium. Nature 488,    680 (Aug. 30, 2012).-   14. S. Sharma et al., Innate immune recognition of an AT-rich    stem-loop DNA motif in the Plasmodium falciparum genome. Immunity    35, 194 (Aug. 26, 2011).-   15. N. Yan, Z. J. Chen, Intrinsic antiviral immunity. Nat Immunol    13, 214 (2012).-   16. V. Pascual, L. Farkas, J. Banchereau, Systemic lupus    erythematosus: all roads lead to type I interferons. Current opinion    in immunology 18, 676 (December, 2006).-   17. Y. Yao, Z. Liu, B. Jallal, N. Shen, L. Ronnblom, Type I    Interferons in Sjogren's Syndrome. Autoimmunity reviews, (Nov. 29,    2012).-   18. R. E. Rigby, A. Leitch, A. P. Jackson, Nucleic acid-mediated    inflammatory diseases. Bioessays 30, 833 (September, 2008).-   19. A. Takaoka et al., DAI (DLM-1/ZBP1) is a cytosolic DNA sensor    and an activator of innate immune response. Nature 448, 501 (Jul.    26, 2007).-   20. L. Unterholzner et al., IFI16 is an innate immune sensor for    intracellular DNA. Nature immunology 11, 997 (November, 2010).-   21. Z. Zhang et al., The helicase DDX41 senses intracellular DNA    mediated by the adaptor STING in dendritic cells. Nature immunology    12, 959 (October, 2011).-   22. The GenBank accession numbers for human and mouse cGAS sequences    are KC294566 and KC294567.

Example 3. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages is anEndogenous High Affinity Ligand for STING

Innate immune sensing of microbial infections is mediated bygermline-encoded pattern recognition receptors that include membraneproteins such as Toll-like receptors (TLRs) and cytosolic proteins suchas NOD-like receptors (NLRs) and RIG-I like receptors (RLRs) (Iwasakiand Medzhitov, 2010; Ronald and Beutler, 2010; Takeuchi and Akira,2010). As virtually all infectious microorganisms contain and neednucleic acids in their life cycles, the innate immune system has evolvedto recognize the microbial DNA and RNA as a central strategy of hostdefense. Specifically, several TLRs are localized on the endosomalmembrane to detect RNA or DNA in the lumen of the endosomes, whereasRLRs are responsible for detecting viral and bacterial RNA in thecytoplasm.

DNA is known to be an immune stimulatory molecule for more than acentury, but how DNA activates the host immune system has not beenextensively investigated until recently (O'Neill, 2013). DNA in theendosome is detected by TLR9, which then triggers the production oftype-I interferons and inflammatory cytokines. When microbial or hostDNA is delivered to the cytoplasm, it can also induce type-I interferonsthrough the endoplasmic reticulum membrane protein STING (also known asMITA, ERIS or MPYS) (Barber, 2011). STING functions as an adaptorprotein that recruits and activates the protein kinases IKK and TBK1,which in turn activate the transcription factors NF-κB and IRF3 toinduce interferons and other cytokines.

We recently identified cyclic GMP-AMP Synthase (cGAS) as a DNA sensorthat activates STING (Sun et al., 2013; Wu et al., 2013). Specifically,we found that cGAS catalyzes the synthesis of cyclic GMP-AMP (cGAMP)from ATP and GTP in the presence of DNA. cGAMP then functions as asecond messenger that binds to and activates STING. While these studiesclearly demonstrate that cGAMP is an endogenous second messengerproduced by cGAS in mammalian cells, the exact nature of the internalphosphodiester linkages between GMP and AMP in cGAMP was not determinedin part because mass spectrometry alone could not unambiguouslydistinguish these linkages without the availability of all cGAMP isomersas the standard reference. Although chemically synthesized cGAMP thatcontains homogenous 3′-5′ linkages is capable of inducing IFNβ, itremained possible that cGAMP containing other phosphodiester linkagesmight also activate the STING pathway.

In this study, we further investigated the structure of cGAMP through acombination of chemical and biophysical techniques. We found that cGAMPproduced by cGAS contains a phosphodiester linkage between 2′-OH of GMPand 5′-phosphate of AMP and another between 3′-OH of AMP and5′-phosphate of GMP. We further showed that this molecule, hereinreferred to as 2′3′-cGAMP, was produced in mammalian cells in responseto DNA in the cytoplasm. Moreover, we demonstrated that 2′3′-cGAMP bindsto STING with a high affinity and is a potent inducer of interferon-0(IFNβ). We also solved the crystal structure of STING bound to the cGASproduct and observed extensive interactions between 2′3′-cGAMP andSTING, which provide the structural basis for their specific and highaffinity binding. Importantly, the structure of the STING—cGAMP complexrevealed that this natural ligand induces conformational rearrangementsin STING underlying its activation.

The Product of cGAS is Cyclic GMP-AMP Containing Mixed PhosphodiesterBonds.

Both 2′-5′ and 3′-5′ phosphodiester linkages between nucleotides areknown to exist in nature while the 2′-5′ linkage is less common. Theinternal phosphodiester linkages of the natural cGAMP produced by cGASremain to be determined. We therefore chemically synthesized cGAMPmolecules containing all four possible phosphodiester linkages (TableSi). The chemical synthesis of cGAMP isoforms was performed usingprocedures modified from published methods (Gaffney et al., 2010; Zhanget al., 2006). For simplicity, we name these cGAMP molecules accordingto the OH position of GMP followed by the OH position of AMP that formthe phosphodiester bonds; for example, 2′3′-cGAMP contains aphosphodiester linkage between 2′-OH of GMP and 5′-phosphate of AMP andanother between 3′-OH of AMP and 5′-phosphate of GMP. We also usedpurified cGAS protein to enzymatically synthesize the natural cGAMP fromATP and GTP in the presence of DNA (Sun et al., 2013). The purified cGASproduct and synthetic cGAMP isomers were analyzed by nuclear magneticresonance (NMR) spectroscopy. Strikingly, the ¹H NMR spectrum of thecGAS product was identical to that of synthetic 2′3′-cGAMP, but distinctfrom those of other cGAMP isomers. In particular, the anomeric proton(H1′) was a singlet with a 3′-phosphate and a doublet with 2′-phosphate.Consistently, only the phosphates of 2′,3′-cGAMP had the same ³¹P NMRchemical shifts as those of natural cGAMP. We also performed massspectrometry analysis of the natural and synthetic cGAMP usingQ-Exactive, an instrument with high resolution and mass accuracy. Thetotal mass of each of these singly charged molecules ([M+H]⁺) was675.107, exactly matching the theoretical mass of cGAMP. The tandem mass(MS/MS) spectra of the cGAS product, which was fragmented using higherenergy collision dissociation (HCD), were identical to those ofsynthetic 2′3′-cGAMP, and similar but not identical to those of2′2′-cGAMP and 3′3′-cGAMP. The MS/MS spectra of 3′2′-cGAMP appeared tobe most distinct from those of 2′3′-cGAMP and the cGAS product. Reversephase HPLC analysis showed that natural cGAMP co-eluted with 2′3′-cGAMP,but not other cGAMP molecules. We also determined the configuration ofthe cGAS product by circular dichroism (CD), confirming that it isderived from D-ribose. The CD spectrum of the natural cGAMP overlappedwell with that of 2′3′-cGAMP. The near-UV CD spectra indicate that thefour cGAMPs adopt significantly different conformations, with 2′3′ and2′2′-cGAMPs forming a CD band pattern distinct from those of 3′2′- and3′3′-cGAMPs. Collectively, these results provide definitive proof thatcGAS synthesizes 2′3′-cGAMP in vitro.

Endogenous cGAMP Produced in DNA-Transfected Cells Contains MixedPhosphodiester Bonds.

To test whether mammalian cells could produce endogenous cGAMP thatcontains the mixed phosphodiester linkages, we transfected the mousecell line L929 and human monocytes THP1 with herring testis DNA(HT-DNA), then cell lysates were heated at 95° C. to denature proteinsand the supernatants were prepared for analysis of endogenous cGAMP bymass spectrometry (Wu et al., 2013). The MS/MS spectra of the endogenousmolecule from both cell lines were identical to those of cGAS productand 2′3′-cGAMP, indicating that the endogenous second messenger is2′3′-cGAMP.

2′3′-cGAMP is a High Affinity Ligand of STING.

We performed isothermal titration calorimetry experiments to measure theaffinity (K_(d)) of STING binding to natural and synthetic cGAMP. AC-terminal domain (CTD) encompassing residues 139-379 of human STING,which was previously shown to mediate binding to the bacterial secondmessenger cyclic di-GMP (Burdette et al., 2011; Huang et al., 2012;Ouyang et al., 2012; Shang et al., 2012; Shu et al., 2012; Yin et al.,2012), was expressed in E. coli and purified to apparent homogeneity forthe ITC experiment. Consistent with previous reports, we found thatc-di-GMP bound to STING with a K_(d) of 1.21 uM. Interestingly, bothnatural cGAMP and synthetic 2′3′-cGAMP bound to STING with such a highaffinity that curve fitting was difficult. In addition, unlike thebinding of c-di-GMP, which is an exothermic process, the binding ofnatural and 2′3′-cGAMP to STING was endothermic, suggesting that theenergy may be used for STING conformational change (see below). Toobtain the K_(d) of natural and synthetic 2′3′-cGAMP for STING, wetitrated different amounts of these compounds as competitors into theSTING—c-di-GMP complex. These measurements yielded a K_(d) of 4.59 nMfor the cGAS product and 3.79 nM for 2′3′-cGAMP. The competitionexperiment was also performed for 3′2′-cGAMP, because its binding toSTING generated little heat change. This compound binds to STING with aK_(d) of 1.61 uM. 2′2′- and 3′3′-cGAMP were titrated directly to STINGand the K_(d) values were calculated to be 287 nM and 1.04 uM,respectively. Thus, the K_(d) of 2′3′-cGAMP was ˜300 fold lower thanthose of c-di-GMP, 3′2′-cGAMP and 3′3′-cGAMP, and ˜75 fold lower thanthat of 2′2′-cGAMP.

cGAMPs are Potent Inducers of Type-I Interferons.

We delivered different amounts of the cGAMP isomers as well as c-di-GMPinto L929 cells and measured IFN3 induction by q-RT-PCR. The cGAMPmolecules induced IFN (with an EC₅₀ that ranged from 15 nM to 42 nM,whereas c-di-GMP had an EC₅₀ of greater than 500 nM. Thus, it appearedthat the binding affinity of different cyclic di-nucleotides did notcorrelate well with their EC₅₀ in the cell-based assays. The reason forthis is not clear, but it is possible that different compounds havedifferent stability or distribution in the cells. Nevertheless, theseexperiments provide direct evidence that the cGAS product, 2′3′-cGAMP,is a high affinity ligand for STING (K_(d): ˜4 nM) and a potent inducerof IFNβ in cells (EC₅₀: ˜20 nM).

The Crystal Structure of STING-cGAMP Complex Reveals Ligand-InducedConformational Rearrangements of STING.

We co-crystallized the STING C-terminal domain (CTD) (residues 139-379)with the purified cGAS product in the C2 space group. The structure ofthe complex was solved by molecular replacement using an apo-STINGstructure (PDB code: 4F9E) as the search model and was refined to 1.88 Aresolution (Table M1). There is one STING protomer in thecrystallographic asymmetric unit, which forms a butterfly-shaped dimerwith another protomer that is related by the crystallographic two-foldsymmetry. The bound cGAMP molecule sits at the two-fold axis (seedetails below). The ordered region of STING (from Asn152 to Glu336)adopts an overall structure similar to the apo-STING, characterized by acentral twisted 0 sheet surrounded by four α helices. However, STING incomplex with cGAMP displays several striking differences from apo-STINGin both the structure of the monomer and the arrangement of the dimer.Compared with the apo-dimer, the two protomers in the dimer of thecomplex structure undergo substantial inward rotations in relation tothe cGAMP binding site. This more closed arrangement creates a deeperpocket between the two protomers to embrace cGAMP. In addition, thecGAMP binding site is covered by a lid of four-stranded anti-parallelβ-sheet and the connecting loops formed by residues 219-249 from each ofthe two protomers. In contrast, this segment in the apo-structure islargely disordered (Ouyang et al., 2012; Yin et al., 2012). Theformation of the 3 sheet is not due to crystallographic packing. Theinterdomain interactions within the lid involve several pairs of polarcontacts, between the side group of Tyr245 and the main-chain carbonyloxygen atom of Gly234, the side group of Ser243 and the main-chain amidenitrogen atom of Lys236, as well as the side groups of Asp237 andLys224.

Extensive Interactions Between 2′3′-cGAMP and STING Underlie theirSpecific and High Affinity Binding.

Since the crystallographic two-fold axis passes through the asymmetric2′3′-cGAMP molecule, cGAMP must adopt two orientations related by thetwo-fold symmetry. This is consistent with the fact that the twoprotomers in the STING dimer are expected to have equal probabilities tointeract with either the guanidine or the adenosine moiety. We thereforeassigned two alternative conformations with the occupancy of 0.5 forcGAMP and several surrounding amino acid residues. Simulated annealingomit map of the refined structure shows decent density for cGAMP.2′3′-cGAMP, but not other isoforms, fits the electron density map well.Compared to c-di-GMP bound to STING, cGAMP sits ˜2.5 Å deeper in thecrevice between the STING dimeric interface. In addition, the two wingsof the butterfly are ˜20 Å closer to each other in the STING:cGAMPstructure due to the more closed arrangement of the two STING protomers.Further analyses of the cGAMP binding pocket show that cGAMP is wellcoordinated by extensive polar and hydrophobic interactions. The ringsof cGAMP purine base groups stack against four around aromatic residues,Tyr 240 and Tyr167 from each of the two protomers. Notably, the twoa-phosphate groups of cGAMP contact Arg238 from both of the twoprotomers and Arg232 from one protomer. The free 3′-OH of GMP points totwo Ser162 residues from the lower part of the pocket. The guanine basedirectly interacts with the side groups of Glu260 and Thr263, as well asthe main-chain carbonyl oxygen of Val239. These unique polar contactsexplain why 2′3′-cGAMP is a specific and high affinity ligand for STING.Besides, residues from the β-sheet (Arg232, Arg238, Val239), which areinvolved in the cGAMP binding, are likely to control the formation ofthe lid and further activation of STING.

Arginine 232 of STING is important for the cytosolic DNA signalingpathway.

Three previous reports of the crystal structures of STING bound tocyclic-di-GMP used a rare human variant that substitutes Arg232 with ahistidine (Ouyang et al., 2012; Shu et al., 2012; Yin et al., 2012).Extensive sequencing of DNA from human populations has shown that theArg232 allele is prevalent and thus should be considered wild-type STING(Jin et al., 2011). The use of the H232 variant of STING may explain whyc-di-GMP did not induce a significant conformational change of STING inthese studies (Ouyang et al., 2012; Shu et al., 2012; Yin et al., 2012).A previous report showed that a mutation of Arg231 of mouse STING(equivalent to Arg232 in human STING) to alanine abolished IFNβinduction by cyclic-di-GMP, but not DNA (Burdette et al., 2011).However, based on our crystal structure of the STING-cGAMP complex, amutation of Arg232 to histidine is expected to significantly weakencGAMP binding and downstream signaling by STING, and a mutation ofArg232 to alanine should be even more detrimental. We thereforeinvestigated the function of Arg232 of STING in two sets of experiments.First, we knocked down endogenous STING by RNAi in L929 cells andreplaced it with WT, R232A or R232H of human STING. These stable celllines were transfected with HT-DNA or treated with 2′3′-cGAMP, followedby measurement of IFNβ by q-RT-PCR. The cells expressing WT STING wereable to induce IFNβ in response to DNA or cGAMP stimulation, whereasthose expressing either R232A or R232H were defective. As a control, thedouble stranded RNA analogue poly[I:C] stimulated IFNβ expression in allof these cell lines. Second, we stably expressed WT or mutant STING inHEK293T cells, which have undetectable expression of endogenous STINGand cGAS (Sun et al., 2013). The cells were then transfected with thehuman cGAS expression plasmid followed by measurement of IFNβ RNA. WTSTING, but not the R232A mutant, was able to support IFNβ induction bycGAS. The R232H mutant was partially defective, possibly because thepositively charged histidine may weakly substitute for some of thefunctions of Arg232. MAVS, an essential adaptor protein of the RIG-Ipathway (Seth et al., 2005), was able to induce IFNβ in all of thesecell lines. Taken together, our structural and functional data stronglyindicate an important role of Arg232 in the functions of STING andfurther underscore the role of cGAS as an indispensable cytosolic DNAsensor.

Discussion.

Our previous studies identified cGAS as a cytosolic DNA sensor and acyclase that synthesizes cGAMP using ATP and GTP as the substrates (Sunet al., 2013; Wu et al., 2013). cGAMP then functions as a secondmessenger that binds to and activates STING. Here we employed chemicalsynthesis and several biophysical approaches to further characterize theinternal phosphodiester linkages of the cGAS product and determined thatit is 2′3′-cGAMP. Subsequently, Gao et al reported the structures ofcGAS in its apo- and DNA-bound forms, which confirmed that cGAS isindeed a DNA-activated cyclic-GMP-AMP synthase that catalyzes thesynthesis of cGAMP from ATP and GTP (Gao et al., 2013). This elegantstudy also elucidated the structural mechanism by which DNA bindingleads to the activation of cGAS. Using a different approach, Gao et alalso found that the truncated cGAS protein synthesizes 2′3′-cGAMP invitro. However, they did not test whether 2′3′-cGAMP has any biologicalor biochemical activity, nor did they show whether endogenous 2′3-cGAMPis produced in mammalian cells. In this report, we show that stimulationof mouse and human cells with DNA leads to the production of endogenous2′3′-cGAMP. Moreover, we demonstrate that 2′3′-cGAMP binds to STING witha much greater affinity than other cGAMP isomers and c-di-GMP. Wefurther show that 2′3′-cGAMP and other cGAMP isomers are much morepotent than c-di-GMP in inducing IFNβ in cells.

Further insights into the structure and function of 2′3′-cGAMP aregained from the crystal structure of the STING CTD bound to thisendogenous ligand. This crystal structure has a resolution of 1.88A,allowing for a detailed view of the ligand structure, including both2′-5′ and 3′-5′phosphodiester linkages. The structure reveals specificresidues on STING that mediate the binding of 2′3′-cGAMP. Furthermore, acomparison of this structure to the previously published STING CTDstructures in its apo form reveals extensive conformationalrearrangements induced by the natural ligand. Specifically, the two armsof the V shaped STING dimer move closer by about 20 A and a new fourβ-stranded sheet forms a lid above the cGAMP binding site in theligand-bound STING structure. These features are absent in thepreviously determined STING:c-di-GMP structures, which used a STINGvariant containing the R232H mutation. In these structures, c-di-GMPbinding does not induce any obvious conformational rearrangement inSTING (Ouyang et al., 2012; Shu et al., 2012; Yin et al., 2012).However, in two other structures containing the WT STING (Arg232) andc-di-GMP, one exhibits similar conformational changes as observed in theSTING-cGAMP complex (Huang et al., 2012), and the other shows a distinctconformational change in that Arg232 is oriented differently (Shang etal., 2012). The “closed” conformation observed by Huang et al may havecaptured the active state of STING induced by c-di-GMP, which is capableof activating STING, albeit more weakly than cGAMP.

The extensive interactions between STING and 2′3′-cGAMP provide thestructural basis for their high affinity binding. In particular, Glu260,Thr263 and Val239 interact with the guanine base of GMP and Ser162interacts with the free 3′—OH group of GMP, explaining why cGAMPcontaining a phosphodiester bond between 2′-OH of GMP and 5′-phosphateof AMP is a high affinity ligand. In addition, the two a-phosphategroups interact with Arg232 from one protomer and Arg238 from bothprotomers. This structural analysis explains that the R232A or R232Hmutations strongly impair the function of STING in response to DNA orcGAMP. Our data highlight the importance of using the wild-type (Arg232)STING in structural and functional studies.

Although 2′3-cGAMP binds to STING with a much higher affinity than cGAMPisomers containing other phosphodiester linkages, all four cGAMP isomersinduced IFNβ with similar EC₅₀ values, which were much lower than thatof c-di-GMP. Thus, all cGAMP isoforms are potent inducers of IFNβ.

In summary, our results demonstrate that 1) the endogenous secondmessenger produced in mammalian cells in response to cytosolic DNAstimulation is 2′3′-cGAMP; 2) 2′3′-cGAMP is a high affinity ligand forSTING; 3) 2′3′-cGAMP is a potent inducer of IFNβ in mammalian cells; 4)2′3′-cGAMP induces conformational rearrangements in STING that mightunderlie its activation; and 5) extensive interactions between2′3′-cGAMP and STING observed in the crystal structure of the complexexplains their specific and high affinity binding.

We conclude: 2′3′-cGAMP is an endogenous second messenger produced bymammalian cells; 2′3′-cGAMP is a high affinity ligand for STING;2′3′-cGAMP is a potent inducer of type-I interferons; and 2′3′-cGAMPbinding induces conformational changes of STING.

Accession Number.

The coordinates of 2′3′-cGAMP bound human STING CTD structure have beendeposited in the RCSB protein data bank (PDB: 4KSY).

REFERENCES

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TABLE M1 Statistics of data collection and refinement of cGAMP boundSTING Data cGAMP bound STING Space Group C2 Unit Cell (Å, °) 89.52577.927 35.974 90 96.98 90 Number of molecules in ASU 1 Wavelength (Å)0.97918 Resolution (Å)   50-1.88 (1.91-1.88) R_(merge) (%)  7.8 (65.0)I/σ 17.82 (2.20)  Completeness (%) 99.4 (98.6) Number of measuredreflections 99,635 Number of unique reflections 19,800 Redundancy 5.0(4.8) Wilson B factor (Å²) 30.80 R-factor (%) 16.07 (23.09) R_(free) (%)18.15 (30.83) Number of atoms Macromolecules 1483 Ligand 45 Water 72 Allatoms 1600 Average B value (Å²) Macromolecules 46.20 Ligand 23.10solvent 50.00 All atoms 45.70 Rms deviations from ideal values Bonds (Å)0.007 Angle (°) 1.213 Ramachandran plot statistics (%) Favored 97.22Allowed 2.78 Outliers 0 Values in parentheses are for the highestresolution shell. R = Σ|F_(obs) − F_(calc)|/ΣF_(obs), where Fcalc is thecalculated protein structure factor from the atomic model (Rfree wascalculated with 10% of the reflections selected).

TABLE S1 Chemical synthesis of cGAMPs.

(A) Structure of building blocks S1-S4.

(B) Synthesis of building blocks S1 and S2.

(C) Synthesis of building blocks S3 and S4.

(D) Synthesis of 2′3′-cGAMP.

(E) Synthesis of 2′2′-cGAMP.

(F) Synthesis of 3′2′-cGAMP.

(G) Synthesis of 3′3′-cGAMP.

Example 4. Cyclic GMP-AMP Synthase is an Innate Immune Sensor of HIV andOther Retroviruses

Retroviruses, including HIV, can activate innate immune responses, butthe host sensors for retroviruses are largely unknown. Here we show thatHIV infection activates cyclic-GMP-AMP (cGAMP) synthase (cGAS) toproduce cGAMP, which binds to and activates the adaptor protein STING toinduce type-I interferons and other cytokines. Inhibitors of HIV reversetranscriptase, but not integrase, abrogated interferon-β induction bythe virus, indicating that the reverse transcribed HIV DNA triggers theinnate immune response. Knockout or knockdown of cGAS in mouse or humancell lines blocked cytokine induction by HIV, murine leukemia virus(MLV) and Simian immunodeficiency virus (SIV). These results indicatethat cGAS detects retroviral DNA and that cGAS is an innate immunesensor of HIV and other retroviruses.

Although tremendous advances have been made in our understanding ofinnate immune recognition of many microbial pathogens (1-3), relativelylittle is known about innate immune responses against retroviralinfections (4). Retroviruses were thought to trigger weak or no innateimmune responses, which were typically measured through the productionof inflammatory cytokines and type-I interferons. However, recentresearch has shown that retroviruses such as HIV can trigger innateimmune responses, which are normally masked by viral or host factors(5-8). For example, TREX1 is a cytosolic exonuclease that degrades DNAderived from HIV or endogenous retroelements, thereby preventing theaccumulation of cytosolic DNA which would otherwise trigger innateimmunity (9, 10). Loss of function mutations of TREX1 in humans havebeen closely linked to Aicardi Goutieres Syndrome (AGS), a lupus-likedisease characterized by elevated expression of inflammatory cytokinesand interferon-stimulated genes (11).

We have recently identified the enzyme cyclic GMP-AMP (cGAMP) synthase(cGAS) as a cytosolic DNA sensor that triggers the production of type-Iinterferons and other cytokines (12, 13). DNA binds and activates cGAS,which catalyzes the synthesis of a unique cGAMP isomer from ATP and GTP.This cGAMP isomer, termed 2′3′-cGAMP, which contains both 2′-5′ and3′-5′ phosphodiester linkages, functions as a second messenger thatbinds and activates the endoplasmic reticulum protein STING (14-17).STING then activates the protein kinases IKK and TBK1, which in turnactivate the transcription factors NF-κB and IRF3 to induce interferonsand other cytokines (18). Knockdown of cGAS inhibits IFNβ induction byDNA viruses such as herpes simplex virus-1 (HSV-1) and vaccinia virus(13). Because retroviruses generate complementary DNA from the viral RNAby reverse transcription, we hypothesized that cGAS might detectretroviral DNA and trigger innate immune responses.

We used a single-round HIV-1 virus in which its envelope protein wasreplaced with the glycoprotein of vesicular stomatitis virus (VSV-G),which allows it to infect a large variety of human and mouse cell types(9). This virus also expresses GFP, which can be used to monitor viralinfection. Infection of the human monocytic cell line THP1 with HIV-GFPled to dimerization of IRF3, a hallmark of its activation.Phosphorylation of STAT1 at Tyr-701 was also detected after HIVinfection, indicating that the interferon signaling pathway wasactivated in the virus infected cells (19). HIV infection led to theinduction of IFNβ and the chemokine CXCL10, concomitant with thegeneration of the HIV Gag episomal DNA. The levels of IFNβ productionwere proportional to the multiplicity of infection by HIV. Treatment ofHIV-GFP virus with DNase I did not impair its ability to induce IFNβ,whereas treatment of herring testis DNA (HT-DNA) with DNase I inhibitedIFNβ induction, indicating that IFNβ induction by HIV-GFP was not due toany contaminating DNA. Differentiation of THP1 from monocytes tomacrophages by treating the cells with phorbol-12-myristate-13-acetate(PMA) inhibited HIV-GFP infection or replication and strongly inhibitedIFNβ induction. Thus, unless otherwise indicated, THP1 cells used in ourstudy were not treated with PMA prior to HIV infection.

To test if reverse transcription is required for HIV to activate theinnate immune response, we treated THP1 cells with the HIV reversetranscriptase inhibitors, azidothymidine (AZT) and nevirapine (NVP).Both inhibitors blocked IRF3 activation and IFNβ induction by HIV. Incontrast, the HIV integrase inhibitor raltegravir (RAL) did not affectthe activation of this pathway. AZT and NVP, even at highconcentrations, did not inhibit IFNβ induction by HT-DNA, indicatingthat the inhibitory effects of AZT and NVP were due to their specificinhibition of HIV reverse transcription. These results indicate that thereverse transcribed HIV DNA is the trigger of IRF3 activation and IFNβproduction.

Strikingly, shRNA-mediated knockdown of cGAS or STING in THP1 cellsstrongly inhibited the induction of IFNβ and CXCL10 and the activationof IRF3 by HIV-GFP. Control experiments showed that shRNA againstluciferase did not inhibit the activation of the pathway, and that theshRNA vectors knocked down the intended targets specifically. Inparticular, the cGAS shRNA knocked down cGAS but not STING, and theinduction of IFNβ in these cells was rescued by delivering cGAMP intothe cells indicating that the cGAS shRNA did not have off-target effectsin the STING pathway.

Previous studies have shown that VSV-G pseudotyped HIV-1 stronglyinduces IFNβ in TREX1-deficient mouse embryonic fibroblasts (MEF) butnot in the wild-type (WT) MEF (9). We generated Trex1^(−/−) MEF celllines stably expressing shRNA against cGAS, STING or luciferase (as acontrol). HIV infection induced IFNβ and CXCL10 RNA in the control cells(sh-luciferase) but not in cGAS or STING depleted cells. In contrast,knockdown of cGAS or STING did not affect the induction of IFNβ orCXCL10 by the double-stranded RNA analogue poly[I:C].

To obtain definitive evidence for the role of cGAS in the innate sensingof cytosolic DNA and retroviruses, we employed the TALEN technology todisrupt the gene that encodes cGAS (Mb21d1), specifically the regionthat encodes the catalytic domain, in L929 cells (20). Although L929cells contain three copies of chromosome 9 that harbors the cGAS gene,DNA sequencing of the TALEN expressing cells identified multiple clonesthat had deletions in all three chromosomes; three of these clones werechosen for further studies. All three clones contained deletions in thecGAS locus that generated frame-shift mutations (21).

All three cGAS mutant cell lines failed to activate IRF3 in response toHT-DNA transfection or herpes simplex virus (HSV-1; a double-strandedDNA virus) infection. As controls, these cells activated IRF3 normallyin response to transfection with poly[I:C] or infection with Sendaivirus, an RNA virus. The cGAS mutant cells were also defective ininducing CXCL10 in response to HT-DNA, but this defect was rescued bytransfecting the cells with the mouse cGAS expression plasmid.

We chose cGAS mutant clone #18 and the parental L929 cells toinvestigate the role of cGAS in innate immune recognition of HIVinfection. In L929 cells stably expressing an shRNA against TREX1, butnot the control luciferase, HIV-GFP infection induced IRF3 dimerizationand the production of IFNβ and CXCL10. In contrast, the L929 cGAS mutantcells failed to mount any detectable immune response to HIV infectioneven when TREX1 was depleted, demonstrating the essential role of cGASin immune responses against HIV. The depletion of cGAS did not affectIFNβ or CXCL10 induction by Sendai virus.

We have previously shown that HEK293T cells do not express detectablelevels of cGAS and STING and thus fail to activate IRF3 in response toDNA transfection or DNA virus infection (13). Consistent with animportant role of cGAS and STING in retrovirus detection, HIV-GFPinfection activated IRF3 and STAT1 in THP1 but not HEK293T cells. Incontrast, Sendai virus activated IRF3 and STAT1 in both cell lines. Todetermine if HIV infection leads to the production of endogenous cGAMPin human cells, we prepared lysates from HIV-infected THP1 and HEK293Tcells, heated the lysates at 95° C. to denature most proteins, whichwere removed by centrifugation (12). The supernatant that potentiallycontained cGAMP was delivered to THP1 cells that had been permeabilizedwith the bacterial toxin perfringolysin-O (PFO), and then IRF3dimerization was assayed by native gel electrophoresis. Theheat-resistant supernatant from HIV-infected THP1, but not HEK293Tcells, contained the cGAMP activity that stimulated IRF3 activation inthe recipient cells. Furthermore, inhibition of HIV reversetranscription by AZT, DDI (didanosine) or NVP blocked the generation ofthe cGAMP activity, whereas the HIV integrase inhibitor RAL had noeffect. HIV-GFP infection in L929-shTrex1 cells also led to generationof the cGAMP activity, which was dependent on cGAS. Taken together,these results indicate that HIV infection induces the production ofendogenous cGAMP in a manner that depends on cGAS and reversetranscription of HIV RNA to cDNA.

To test if HIV infection produces retroviral cDNA in the cytoplasm toactivate cGAS, we infected HEK293T cells with HIV-GFP and preparedcytosolic extracts that were then incubated with purified cGAS proteinin the presence of ATP and GTP. Cytosolic extracts from HIV-infectedcells, but not from uninfected cells, were able to stimulate cGAS toproduce the cGAMP activity that activated IRF3 in permeabilized THP1cells. Treatment of HEK293T cells with AZT inhibited the generation ofthe cGAS stimulatory activity. Further analyses showed that thecytoplasm of HIV-infected cells contained the HIV Gag DNA and GFPprotein, both of which were inhibited by AZT.

Quantitative measurement of cGAMP abundance by mass spectrometry usingselective reaction monitoring (SRM) provided the direct evidence thatcGAMP was produced in HIV-infected, but not mock-treated, THP1 cells.Tandem mass spectrometry of the endogenous cGAMP from HIV-infected THP1cells revealed that it was identical to the cGAS product, 2′3′-cGAMP(15).

To test whether HIV infection in primary human immune cells leads tocGAMP production, we infected monocyte-derived macrophages (MDM) andmonocyte-derived dendritic cells (MDDC) with the clinical HIV-1 isolateHIV-BaL and HIV-GFP, respectively. Previous research has shown thathuman macrophages and dendritic cells express SAMHD1, a nuclease thathydrolyzes dNTP, thereby inhibiting HIV reverse transcription. HIV-2 andsimian immunodeficiency virus (SIV) contain the protein Vpx, whichtargets SAMHD1 for ubiquitin-mediated proteasomal degradation, thusremoving this host restriction factor. To facilitate HIV infections inhuman MDMs and MDDCs, we delivered the SIV Vpx into these cells using avirus-like particle (VLP) before HIV infection. In the presence of Vpx,infection of MDMs and MDDCs with HIV-BaL and HIV-GFP, respectively, ledto the generation of cGAMP activity. Quantitative mass spectrometryanalysis further confirmed the production of 2′3′-cGAMP in HIV-infectedMDDCs that expressed Vpx. The cGAMP activity was consistently observedin MDDCs and MDMs of additional human donors, and this activity washigher in the cells infected with HIV than those treated with Vpx alone.These results demonstrate that HIV infection in human macrophages anddendritic cells lead to the generation of cGAMP under conditions thatare permissive to viral replication.

Finally, we tested whether cGAS is required for innate immune responsesagainst other retroviruses by infecting L929 and L929-cGAS KO cell lineswith murine leukemia virus (MLV) and SIV. Similar to HIV, MLV and SIVinduced IFNβ and CXCL10 RNA in L929 cells depleted of endogenous TREX1,but such induction was completely abolished in the cGAS KO cells. Infurther support of an essential role of the cGAS-STING pathway in innateimmune sensing of retroviruses, knockdown of cGAS or STING inTrex1^(−/−) MEF cells strongly inhibited IFNβ induction by MLV and SIV.

Here we demonstrate that cGAS is essential for innate immune responsesagainst HIV, SIV and MLV, indicating that cGAS is a general innateimmune sensor of retroviral DNA. Although HIV primarily infects humanCD4 T cells, it can also enter macrophages and dendritic cells, normallywithout triggering an overt innate immune response by concealing theviral nucleic acids within the capsid and by limiting the accumulationof viral DNA through co-opting host factors such as TREX1 and SAMHD1(8). The absence of a rigorous innate immune response to HIV indendritic cells is thought to be a major factor that hampers productiveT cell responses and vaccine development (7). Our finding that HIV andother retroviruses can induce the production of cGAMP through cGAS underpermissive conditions indicates that cGAMP can be used to bypass theblock of innate immune responses against HIV. As such, cGAMP provides auseful vaccine adjuvant for HIV and other pathogens that are adept atsubverting the host innate immune system.

REFERENCES AND NOTES

-   1. A. Iwasaki, R. Medzhitov, Regulation of adaptive immunity by the    innate immune system. Science 327, 291 (Jan. 15, 2010).-   2. O. Takeuchi, S. Akira, Pattern recognition receptors and    inflammation. Cell 140, 805 (Mar. 19, 2010).-   3. P. C. Ronald, B. Beutler, Plant and animal sensors of conserved    microbial signatures. Science 330, 1061 (Nov. 19, 2010).-   4. R. Medzhitov, D. Littman, HIV immunology needs a new direction.    Nature 455, 591 (Oct. 2, 2008).-   5. N. Manel, D. R. Littman, Hiding in plain sight: how HIV evades    innate immune responses. Cell 147, 271 (Oct. 14, 2011).-   6. N. Manel et al., A cryptic sensor for HIV-1 activates antiviral    innate immunity in dendritic cells. Nature 467, 214 (Sep. 9, 2010).-   7. J. Luban, Innate immune sensing of HIV-1 by dendritic cells. Cell    host & microbe 12, 408 (Oct. 18, 2012).-   8. N. Yan, Z. J. Chen, Intrinsic antiviral immunity. Nat Immunol 13,    214 (2012).-   9. N. Yan, A. D. Regalado-Magdos, B. Stiggelbout, M. A.    Lee-Kirsch, J. Lieberman, The cytosolic exonuclease TREX1 inhibits    the innate immune response to human immunodeficiency virus type 1.    Nature immunology 11, 1005 (November, 2010).-   10. D. B. Stetson, J. S. Ko, T. Heidmann, R. Medzhitov, Trex1    prevents cell-intrinsic initiation of autoimmunity. Cell 134, 587    (Aug. 22, 2008).-   11. Y. J. Crow et al., Mutations in the gene encoding the 3′-5′ DNA    exonuclease TREX1 cause Aicardi-Goutieres syndrome at the AGS1    locus. Nature genetics 38, 917 (August, 2006).-   12. J. Wu et al., Cyclic GMP-AMP is an endogenous second messenger    in innate immune signaling by cytosolic DNA. Science 339, 826 (Feb.    15, 2013).-   13. L. Sun, J. Wu, F. Du, X. Chen, Z. J. Chen, Cyclic GMP-AMP    synthase is a cytosolic DNA sensor that activates the type I    interferon pathway. Science 339, 786 (Feb. 15, 2013).-   14. P. Gao et al., Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan    Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase.    Cell 153, 1094 (May 23, 2013).-   15. X. Zhang et al., Cyclic GMP-AMP Containing Mixed Phosphodiester    Linkages Is An Endogenous High-Affinity Ligand for STING. Molecular    cell, (Jun. 3, 2013).-   16. E. J. Diner et al., The Innate Immune DNA Sensor cGAS Produces a    Noncanonical Cyclic Dinucleotide that Activates Human STING. Cell    Rep 3, 1355 (May 30, 2013).-   17. A. Ablasser et al., cGAS produces a 2′-5′-linked cyclic    dinucleotide second messenger that activates STING. Nature 498, 380    (Jun. 20, 2013).-   18. G. N. Barber, Cytoplasmic DNA innate immune pathways.    Immunological reviews 243, 99 (September, 2011).-   19. D. E. Levy, J. E. Damell, Jr., Stats: transcriptional control    and biological impact. Nature reviews. Molecular cell biology 3, 651    (September, 2002).-   20. T. Cermak et al., Efficient design and assembly of custom TALEN    and other TAL effector-based constructs for DNA targeting. Nucleic    Acids Res 39, e82 (July, 2011).-   21. Clone #18 has frame-shift mutations in all three chromosomes. In    addition to frame-shifts, clone #36 harbored a 9-bp deletion in one    chromosome that removed 3 amino acids (215-217) in the catalytic    domain, whereas clone #94 had 12-bp deletion in one chromosome and    18-bp deletion in another that removed 4 (214-217) and 6 (212-217)    amino acids in the catalytic domain, respectively.

Example 5. Pivotal Roles of cGAS-cGAMP Signaling in Antiviral Defenseand Immune Adjuvant Effects

Invasion of microbial DNA into the cytoplasm of animal cells triggers acascade of host immune reactions that help clear the infection; however,self DNA in the cytoplasm can cause autoimmune diseases. Biochemicalapproaches led to the identification of cyclic GMP-AMP (cGAMP) synthase(cGAS) as a cytosolic DNA sensor that triggers innate immune responses.Here we show that cells from cGAS-deficient (cGas^(−/−)) mice, includingfibroblasts, macrophages and dendritic cells, failed to produce type-Iinterferons and other cytokines in response to DNA transfection or DNAvirus infection. cGas^(−/−) mice were more susceptible to lethalinfection with herpes simplex virus-1 (HSV1) than wild type mice. Wealso show that cGAMP is an adjuvant that boosts antigen-specific T cellactivation and antibody production.

The detection of foreign DNA invasion is a fundamental mechanism of hostdefense. In mammalian cells, the presence of foreign or self DNA in thecytoplasm is a danger signal that triggers the host innate immuneresponses (1). Through biochemical studies, we have recently identifiedcyclic GMP-AMP (cGAMP) synthase (cGAS) as an innate immune sensor ofcytosolic DNA that triggers the production of type-I interferons andother inflammatory cytokines (2, 3). cGAS binds to DNA independently ofits sequence; this binding activates cGAS to catalyze the synthesis of aunique cGAMP isomer, which contains both 2′-5′ and 3′-5′ phosphodiesterlinkages (4-7). This molecule, termed 2′3′cGAMP, functions as a secondmessenger that binds and activates the adaptor protein STING (3, 7).STING then activates the protein kinases IKK and TBK1, which in turnactivate the transcription factors NF-κB and IRF3 to induce interferonsand cytokines (8).

To investigate the function of cGAS in vivo, we generated a cGasknockout mouse strain, in which the first exon is spliced into a LacZcassette, thus abrogating the expression of the endogenous locus (9).The cGas^(−/−) mice were born at the Mendelian ratio, and did notdisplay any overt developmental abnormality. Quantitative reversetranscription PCR (q-RT-PCR) analyses of RNA from lung fibroblasts andbone marrow derived macrophages (BMDM) confirmed that the cGas^(−/−)cells were defective in producing cGas RNA, whereas cGas^(+/−) cellsproduced intermediate levels of cGas RNA.

We obtained lung fibroblasts from WT, cGas^(+/−) and cGAS^(−/−) mice aswell as the goldenticket (gt/gt) mouse, which has a point mutation thatresults in the loss of expression of STING (10). Transfection ofdifferent types of DNA, including herring testis DNA (HT-DNA), E. coliDNA and interferon stimulatory DNA (ISD; a 45 bp double-stranded DNA)(11), into the lung fibroblasts from WT and cGas^(+/−) mice led torobust production of IFNβ protein, as measured by ELISA. In contrast,the cGas^(−/−) and Sting^(gt/gt) cells failed to produce any detectablelevel of IFNβ. Poly[I:C], a double-stranded RNA analogue known to induceIFNβ through the RIG-I like-receptor (RLR) pathway (12), induced IFNβnormally in the absence of cGas or Sting. Interestingly, poly[dA:dT],which was previously shown to induce type-I interferons through the RNApolymerase III—RIG-I-MAVS pathway (13, 14), induced IFNβ normally in thecGas^(−/−) and Sting^(gt/gt) cells. q-RT-PCR analyses further confirmedthat cGAS is essential for IFNβ RNA induction by different types ofsynthetic or bacterial DNA, except poly[dA:dT]. Time course experimentsshow that IFNβ induction by ISD was completely abolished in cGas^(−/−)lung fibroblasts even at early time points (2-8 hr) after the DNAtransfection, indicating that cGAS is indispensable for IFNβ inductionby cytosolic DNA.

To measure cGAMP production in WT and cGas^(−/−) cells, we performed abioassay that measures the cGAMP activity in cytoplasmic extracts fromISD-transfected cells. The extracts were heated at 95° C. to denaturemost proteins, which were removed by centrifugation. The supernatantsthat might contain cGAMP were delivered to the human monocytic cell lineTHP1, which had been permeabilized with the bacterial toxinperfringolysin-O (PFO). Dimerization of IRF3, a hallmark of itsactivation, was then measured by native gel electrophoresis. This assayshowed that the extracts of ISD-transfected lung fibroblasts from WT butnot cGas^(−/−) mice contained the cGAMP activity, demonstrating thatcGAS has a non-redundant role in catalyzing cGAMP synthesis in thesecells in response to cytosolic DNA.

Next, we infected the lung fibroblasts with the DNA viruses herpessimplex virus-1 (HSV1), vaccinia virus (VACV) and a mutant strain ofHSV1 called d109, which has a deletion of viral proteins such as ICPOthat is known to antagonize immune responses (15). IFNβ induction byeach of these viruses was largely abolished in cGas^(−/−) andSting^(gt/gt) cells, and partially inhibited in cGas^(+/−) cells. Incontrast, IFNβ induction by Sendai virus, an RNA virus known to activatethe RIG-I pathway, was not affected by the deficiency in cGas or Sting.Delivery of cGAMP into the cytoplasm rescued IFNβ induction incGas^(−/−) cells but not Sting^(gt/gt) cells. Similarly, induction ofthe chemokine CXCL10 by the DNA viruses was dependent on cGas and Sting.Measurement of IRF3 dimerization showed that cGas^(−/−) cells failed toactivate IRF3 in response to transfection of HT-DNA or infection by WTHSV1 or the HSV1 strain 7134, which also lacks the interferon antagonistICPO (16). The cGas deficiency did not impair IRF3 activation by Sendaivirus. Thus, cGAS is required for IRF3 activation and cytokine inductionby DNA viruses but not RNA viruses in mouse lung fibroblasts.

BMDM from cGas^(−/−) and Sting^(gt/gt) mice were defective in producingIFNβ in response to transfection with HT-DNA or ISD. Similarly, IFNβinduction by VACV and the HSV1 strains d109 and 7134 was largelyabolished in cGas^(−/−) and Sting^(gt/gt) BMDM. However, IFNβ inductionby WT HSV1 was severely but not completely blocked in either cGas^(−/−)or Sting^(gt/gt)BMDM, indicating that these cells possess anotherpathway that could partially compensate for the loss of the cGAS-STINGpathway to detect WT HSV1 infection. The loss of cGAS or STING in BMDMdid not affect IFNβ induction by Sendai virus. Kinetic experiments showthat IFNβ induction by ISD and HSV1-d109 was abolished in cGas^(−/−)BMDM throughout the time course of stimulation. Similarly to IFNβ, theinduction of TNFα by HT-DNA or ISD was abolished in cGas^(−/−) orStinggtgt BMDM. q-RT-PCR analyses showed that the induction of IFNβ,interleukin-6 (IL6) and CXCL10 RNA by transfection of HT-DNA or ISD orinfection with HSV1-d109 was completely dependent on cGas and Sting. Incontrast, the RNA levels of these cytokines induced by poly[I:C] orSendai virus were not affected by the deficiency in cGas or Sting.

We obtained conventional dendritic cells (cDC) and plasmacytoid DCs(pDC) by culturing bone marrows in conditioned media containing GM-CSFand Flt3 ligand (Flt3L), respectively. The GM-CSF DCs, which containslargely cDC, from the cGas^(−/−) and Sting^(gt/gt) mice failed to induceIFNα or IFNβ in response to transfection of HT-DNA or ISD. The loss ofcGAS or STING in GM-CSF DCs abolished IFNβ induction by HSV1-d109 andVACV, and partially inhibited IFNβ induction by WT HSV1. In contrast,the deficiency in cGAS or STING did not impair IFNα or IFNβ induction bySendai virus. q-RT-PCR experiments further confirmed that cGAS and STINGwere essential for the induction of IFNβ, IL6 and CXCL10 RNA bytransfection with HT-DNA or ISD or infection with HSV1-d109, whereas theinduction of these cytokines by poly[I:C] or Sendai virus wasindependent of cGAS or STING.

pDCs are known to express TLR9 that is responsible for the induction oftype-I interferons by synthetic CpG DNA containing phosphorothioatebonds (17). When the CpG DNA was used to stimulate Flt3L-DCs, whichcontains largely pDCs, in the presence or absence of liposome(lipofectamine 2000), it induced robust production of IFNα and IFNβ evenin the cGas^(−/−) and Sting^(gt/gt) cells. In contrast, other forms ofDNA, including ISD, poly[dA:dT] and genomic DNA from E. coli and Vibriocholerae, induced IFNα in Flt3L-DCs only in the presence of liposome,and this induction by each DNA was abolished in the absence of cGAS orSTING. The strong dependency of IFNα induction by poly[dA:dT] on cGASand STING in pDCs indicates that the cGAS-STING pathway, but not thePol-III-RIG-I pathway, plays a major role in sensing the DNA in thesecells. The Flt3L-DC from the cGas^(−/−) and Sting^(gt/gt) mice inducedIFNα and IFNβ in response to infection by Sendai virus, but not HSV1.Together, these results demonstrate that cGAS is responsible fordetecting natural DNA (e.g., bacterial DNA) and DNA virus infections indendritic cells.

To determine the role of cGAS in immune defense against DNA viruses invivo, we infected WT and cGas^(−/−) mice with HSV1 via the intravenous(i.v) route. ELISA analyses showed that the sera of WT mice containedelevated levels of IFNα and IFNββ, which peaked at 8 and 4 hours,respectively, after HSV1 infection (1×10⁷ pfu/mouse). The levels of IFNαand IFNβ were severely attenuated in the cGas^(−/−) mice infected withthe same infectious dose of HSV1. In an independent experiment in whichthe mice were monitored for their survival after infection with HSV1 atthe infectious dose of 1×10⁶ pfu/mouse, four out of the five cGas^(−/−)mice developed ataxia and paralysis in 3 days after the virus infectionand died a few hours after these symptoms appeared. The fifth cGas^(−/−)mouse died on day 4 after infection. Three out of five WT mice developedthese symptoms on day 6 and died shortly afterwards. When the brains ofWT and cGas^(−/−) mice were extracted to measure viral titers on day 3after infection, high levels of HSV1 were detected in all fivecGas^(−/−) mice, whereas none of the WT mice had detectable levels ofHSV1 in the brains. Similar survival curves were observed and similarviral titers in the brains were detected in independent experimentswhere the infectious dose of HSV1 was increased to 1×10⁷ pfu per mouse.The susceptibility of cGas^(−/−) mice to HSV1 infection was similar tothat of Sting^(gt/gt) mice, which also had marked reduction of IFNα andIFNβ in the sera, and died within 3-4 days after HSV1 infection (18).

Our results that cGAS is essential for the induction of type-Iinterferons by cytosolic DNA in multiple cell types, including antigenpresenting cells, indicate that the cGAS product, 2′3′cGAMP, can be usedto substitute for the immune stimulatory effect of DNA, including theadjuvant effect of DNA vaccines (19). To ascertain the adjuvant effectof 2′3′cGAMP, we injected the model protein antigen ovalbumin (OVA) inthe absence or presence of 2′3′cGAMP into WT or Sting^(gt/gt) mice viathe intramuscular (i.m) route. The mice were boosted once on day 10 withthe same antigen formulation. ELISA analyses showed that 2′3′cGAMPstrongly enhanced the production of OVA-specific antibodies on day 17 inthe WT, but not Sting^(gt/gt) mice. This adjuvant effect of 2′3′cGAMPwas also not observed in type-I interferon receptor deficient mice(Ifnar^(−/−)). To investigate the effect of 2′3′cGAMP on T cellactivation, splenic leukocytes isolated from the WT mice, which had beenimmunized with OVA or OVA+2′3′cGAMP for 7 days, were cultured with anOVA peptide known to stimulate CD4 T cells through the MHC class IImolecule I-A^(b) or another OVA peptide that stimulates CD8 T cellsthrough the MHC class I molecule H-2K^(b). Both CD4 and CD8 T cells fromthe mice immunized with OVA+2′3′cGAMP, but not OVA alone, producedelevated levels of IFNγ and IL-2 after stimulation with the cognatepeptides. Flow cytometry analysis using a tetramer composed of an OVApeptide in complex with H-2K^(b) showed a marked increase in thepercentage of the tetramer-positive CD8 T cells in the mice immunizedwith OVA+2′3′cGAMP, indicating that 2′3′cGAMP stimulated the expansionof CD8 T cells bearing the OVA-specific T cell receptor. Taken together,these results indicate that 2′3′cGAMP functions as an immune adjuvant tostimulate antigen-specific T cell and B cell responses.

Here we provide evidence that cGAS is essential for the induction oftype-I interferons and other inflammatory cytokines by DNA transfectionand DNA virus infection. With the exception of poly[dA:dT] and CpG DNA,most DNA molecules, especially those found in nature (e.g., bacterialand viral DNA), stimulate type-I interferons exclusively through thecGAS-cGAMP-STING pathway. In multiple cell types, including fibroblasts,macrophages and dendritic cells, the induction of type-I interferons byvaccinia viruses and several strains of HSV1 is completely dependent oncGAS and STING. Notably, however, IFNβ induction by wild type HSV1 isseverely but not completely abolished in BMDM and GM-CSF DCs fromcGas^(−/−) or Sting^(gt/gt) mice. Other putative DNA sensors, such asIFI16 or DDX41, may also be involved in this residual induction of IFNβby WT HSV1 (20, 21). In the case of cGAS, the phenotypes of cGas^(−/−)mice are strikingly similar to those of Sting^(−/−) mice (this study andref. 18). These results, together with our biochemical data showing thatcGAS is a cytosolic enzyme activated by its binding to generic DNA (2,3), formally demonstrate that cGAS is a non-redundant and generalcytosolic DNA sensor that activates STING.

We show that 2′3′cGAMP is an effective adjuvant that boosts theproduction of antigen-specific antibodies and T cell responses. Althoughthe bacterial second messengers cyclic di-GMP and cyclic di-AMP arebeing developed as potential vaccine adjuvants (22), 2′3′cGAMP is a muchmore potent ligand of STING than any of the bacterial cyclicdi-nucleotides (7). Thus, 2′3′cGAMP provides a useful adjuvant for nextgeneration vaccines to prevent or treat human diseases, includinginfectious diseases and cancer.

REFERENCES AND NOTES

-   1. L. A. O'Neill, Immunology. Sensing the dark side of DNA. Science    339, 763 (Feb. 15, 2013).-   2. L. Sun, J. Wu, F. Du, X. Chen, Z. J. Chen, Cyclic GMP-AMP    synthase is a cytosolic DNA sensor that activates the type I    interferon pathway. Science 339, 786 (Feb. 15, 2013).-   3. J. Wu et al., Cyclic GMP-AMP is an endogenous second messenger in    innate immune signaling by cytosolic DNA. Science 339, 826 (Feb. 15,    2013).-   4. A. Ablasser et al., cGAS produces a 2′-5′-linked cyclic    dinucleotide second messenger that activates STING. Nature 498, 380    (Jun. 20, 2013).-   5. E. J. Diner et al., The Innate Immune DNA Sensor cGAS Produces a    Noncanonical Cyclic Dinucleotide that Activates Human STING. Cell    Rep 3, 1355 (May 30, 2013).-   6. P. Gao et al., Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second    Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase. Cell    153, 1094 (May 23, 2013).-   7. X. Zhang et al., Cyclic GMP-AMP Containing Mixed Phosphodiester    Linkages Is An Endogenous High-Affinity Ligand for STING. Molecular    cell, (Jun. 3, 2013).-   8. H. Ishikawa, G. N. Barber, The STING pathway and regulation of    innate immune signaling in response to DNA pathogens. Cellular and    molecular life sciences: CMLS 68, 1157 (April, 2011).-   9. cGas^(−/−) mice were generated by in vitro fertilization using    sperms harboring a targeted insertion at the cGas/Mb21d1 locus.-   10. J. D. Sauer et al., The N-ethyl-N-nitrosourea-induced    Goldenticket mouse mutant reveals an essential function of Sting in    the in vivo interferon response to Listeria monocytogenes and cyclic    dinucleotides. Infect Immun 79, 688 (February, 2011).-   11. D. B. Stetson, R. Medzhitov, Recognition of cytosolic DNA    activates an IRF3-dependent innate immune response. Immunity 24, 93    (January, 2006).-   12. M. Yoneyama et al., The RNA helicase RIG-I has an essential    function in double-stranded RNA-induced innate antiviral responses.    Nat Immunol 5, 730 (July, 2004).-   13. A. Ablasser et al., RIG-I-dependent sensing of poly(dA:dT)    through the induction of an RNA polymerase III-transcribed RNA    intermediate. Nat Immunol, (Jul. 16, 2009).-   14. Y. H. Chiu, J. B. Macmillan, Z. J. Chen, RNA polymerase III    detects cytosolic DNA and induces type I interferons through the    RIG-I pathway. Cell 138, 576 (Aug. 7, 2009).-   15. L. A. Samaniego, L. Neiderhiser, N. A. DeLuca, Persistence and    expression of the herpes simplex virus genome in the absence of    immediate-early proteins. Journal of virology 72, 3307 (April,    1998).-   16. G. T. Melroe, N. A. DeLuca, D. M. Knipe, Herpes simplex virus 1    has multiple mechanisms for blocking virus-induced interferon    production. Journal of virology 78, 8411 (August, 2004).-   17. O. Takeuchi, S. Akira, Pattern recognition receptors and    inflammation. Cell 140, 805 (Mar. 19, 2010).-   18. H. Ishikawa, Z. Ma, G. N. Barber, STING regulates intracellular    DNA-mediated, type I interferon-dependent innate immunity. Nature    461, 788 (Oct. 8, 2009).-   19. C. J. Desmet, K. J. Ishii, Nucleic acid sensing at the interface    between innate and adaptive immunity in vaccination. Nature reviews.    Immunology 12, 479 (July, 2012).-   20. Z. Zhang et al., The helicase DDX41 senses intracellular DNA    mediated by the adaptor STING in dendritic cells. Nature immunology    12, 959 (October, 2011).-   21. L. Unterholzner et al., IFI16 is an innate immune sensor for    intracellular DNA. Nature immunology 11, 997 (November, 2010).-   22. W. Chen, R. Kuolee, H. Yan, The potential of 3′,5′-cyclic    diguanylic acid (c-di-GMP) as an effective vaccine adjuvant. Vaccine    28, 3080 (Apr. 19, 2010).

1-15. (canceled)
 16. An injectable pharmaceutical formulation comprisinga pharmaceutically acceptable carrier and a cyclic dinucleotide, whereinthe cyclic dinucleotide comprises a first nucleotide comprising anadenine or guanine moiety, and a second nucleotide comprising an adenineor guanine moiety; and the first and second nucleotides are connected toeach other by 2′-5′ and 3′-5′ bonds, respectively.
 17. Thepharmaceutical formulation of claim 16, wherein the cyclic dinucleotideactivates the STING protein.
 18. The pharmaceutical formulation of claim16, wherein the cyclic dinucleotide is able to bind to the STINGprotein.
 19. The pharmaceutical formulation of claim 18, wherein thecyclic dinucleotide is able to bind to the Y240 residue of the STINGprotein.
 20. The pharmaceutical formulation of claim 18, wherein thecyclic dinucleotide is able to bind to the N242 residue of the STINGprotein.
 21. The pharmaceutical formulation of claim 18, wherein thecyclic dinucleotide is able to bind to the Y240 and N242 residues of theSTING protein.
 22. The pharmaceutical formulation of claim 18, whereinthe first nucleotide comprises a guanine moiety and the secondnucleotide comprises an adenine moiety.
 23. The pharmaceuticalformulation of claim 16, wherein the first nucleotide comprises aguanine moiety and the second nucleotide comprises an adenine moiety.24. The pharmaceutical formulation of claim 16, wherein the formulationis free of other cyclic dinucleotides.
 25. The pharmaceuticalformulation of claim 16, wherein the 2′-5′ and 3′-5′ bonds arephosphodiester bonds.
 26. The pharmaceutical formulation of claim 16,further comprising an immunogen.
 27. The pharmaceutical formulation ofclaim 26, wherein the immunogen is a vaccine.
 28. A method of inducingor promoting an immune response comprising administering by injection toa mammal in need thereof an effective amount of the pharmaceuticalformulation of claim
 16. 29. The method of claim 28, further comprisingadministering an immunogen to the subject.
 30. The method of claim 28,wherein the cyclic dinucleotide activates the STING protein.
 31. Themethod of claim 28, wherein the cyclic dinucleotide is able to bind tothe STING protein.
 32. The method of claim 31, wherein the firstnucleotide comprises a guanine moiety and the second nucleotidecomprises an adenine moiety.
 33. The method of claim 28, wherein thefirst nucleotide comprises a guanine moiety and the second nucleotidecomprises an adenine moiety.
 34. A method of inducing or promoting animmune response by inducing interferon-β (IFNβ), the method comprisingadministering to a mammal in need thereof an effective amount of aformulation comprising a cyclic dinucleotide, wherein the cyclicdinucleotide comprises a first nucleotide comprising an adenine orguanine moiety, and a second nucleotide comprising an adenine or guaninemoiety; and the first and second nucleotides are connected to each otherby 2′-5′ and 3′-5′ bonds, respectively.
 35. The method of claim 34,wherein the cyclic dinucleotide activates the STING protein.
 36. Themethod of claim 34, wherein the cyclic dinucleotide is able to bind tothe STING protein.
 37. The method of claim 36, wherein the cyclicdinucleotide is able to bind to the Y240 residue of the STING protein.38. The method of claim 36, wherein the cyclic dinucleotide is able tobind to the N242 residue of the STING protein.
 39. The method of claim36, wherein the cyclic dinucleotide is able to bind to the Y240 and N242residues of the STING protein.
 40. The method of claim 36, wherein thefirst nucleotide comprises a guanine moiety and the second nucleotidecomprises an adenine moiety.
 41. The method of claim 34, wherein thefirst nucleotide comprises a guanine moiety and the second nucleotidecomprises an adenine moiety.
 42. The method of claim 34, wherein theformulation is free of other cyclic dinucleotides.
 43. The method ofclaim 34, wherein the 2′-5′ and 3′-5′ bonds are phosphodiester bonds.44. The method of claim 34, further comprising administering animmunogen to the subject.
 45. The method of claim 44, wherein theimmunogen is a vaccine.